{"paper_id":"709d0e35-83f7-4028-ac8f-d77efe2a8b86","body_text":"Lung cancer ,  the most common cause of cancer-related mortality worldwide is generally classified into main histological subtypes, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) 1 , 2 . Lung mucosa is constantly exposed to inhaled environmental pollution, dust, smoke, and pathogens; hence a dynamic network of tissue-resident immune cells continually keeps monitoring the lung to maintain tissue homeostasis 3 . The immune system also has a vital role in cancer initiation and progression. Natural killer cells (NK cells) are part of innate immune cells that serve as a first-line of defense with the capacity to eliminate virally infected cells and neoplastic transformations without prior sensitization 4 . These cytotoxic cells mediate antitumor responses by exploiting death receptors, releasing pro-inflammatory cytokines and perforin/granzyme granules exocytosis 5 .\nInterestingly, over 10% of resident lymphocytes in the lung are NK cells 6 . The association of NK cell dysfunction with tumor progression was revealed in KRAS-driven lung cancer, in which NK cells protect tumors at the early stage but cannot prevent tumor progression 7 . Also, it is shown that resident NK cells have a pivotal role in resistance to experimental lung metastasis by producing IFN-γ 8 . Although the infiltration of NK cells was indicated as a favorable prognostic factor in lung cancer 9 , 10 , the functional reactivity of intratumoral NK cells is crucial in addition to the degree of NK cell infiltration. It has been demonstrated that NK cell dysfunction at the late stage is caused by the suppressive tumor microenvironment (TME) 7 . NK cells isolated from NSCLC patients, which depict different expression patterns, have impaired interferon-γ (IFN-γ) production capability and lower cytotoxicity than non-tumoral NK cells 11 – 13 .\nThe activity of NK cells is modulated by a balance between the net input of signals derived from an array of inhibitory and activating receptors 14 . Among these germ-line encoded receptors, killer-cell immunoglobulin-like receptors (KIR) are essential for acquiring NK cell effector function 15 , 16 . KIRs are encoded by a cluster of polymorphic and homologous genes located at chromosomal region 19q13.4 17 . KIR region comprises activating ( 2DS1 - 5 ,  3DS1 ) and inhibitory ( 2DL1 - 3 ,  2DL5A / B ,  3DL1 ) genes, divided by framework KIRs into centromeric ( 3DL3  to  3DP1 ) and telomeric ( 2DL4  to  3DL2 ) intervals 18 . Although (5′) centromeric and (3′) telomeric regions contain various combinations of activating ( aKIR ) and inhibitory ( iKIR ) genes, certain adjacent KIRs tend to be strongly linked 17 , 19 . Considering strong linkage disequilibrium (LD) among  KIR  genes, it is difficult to isolate each  KIR  gene ' s effect on NK cell response 20 . Based on gene content within both intervals, the KIR locus segregates in distinct haplotypes, mainly known as A (fixed combination of genes, mostly  iKIRs ) and B (variable number of  iKIRs / aKIRs ) 17 , 21 . The KIR gene cluster displays tremendous variation due to gene-content diversity and haplotypic variety 17 . In addition to gene content diversity, allelic polymorphism extends KIR variations and accounts for differential expression levels of KIRs on the surface of NK cells 22 , 23 .\nThe classical HLA class I molecules (HLA-A, B, C) as major KIR ligands have been categorized into 4 types of KIR-binding epitopes (C1, C2, Bw4, A3/A11) according to amino acid sequences 24 . KIR2DL1 and KIR2DL2/3 mainly bind to a group of ligands encoded by HLA-C alleles that differed by Lys/ Asn dimorphism at position 80 (HLA-C2 and HLA-C1, respectively) 25 . KIR3DL1 was found to interact with HLA-Bw4, and KIR3DL2 recognizes HLA-A3/A11 26 , 27 . The Ile/Thr dimorphism at position 80 defines the affinity of KIR binding to the Bw4 motif, in which Ile80 exhibits a greater affinity for 3DL1 28 . The growing understanding of activating KIR/ HLA interactions indicates that activating KIR3DS1, 2DS1, and 2DS2 bind to the same HLA class I with lower affinity than their homologous inhibitory counterparts 29 – 32 . However, other KIR/HLA pair interactions haven’t been distinctly demonstrated. It is shown that different iKIRs exhibit various binding affinities for HLA-I ligands. Interestingly the peptide presented by HLA subtypes seems to play a role in binding affinity alteration 33 . Signals derived from iKIRs interacting with self-HLA ligands set a threshold of activation for NK cells leading to a maturation process titled “education” or “licensing” 34 , 35 . Besides development and self-tolerance, education renders NK cells capacity to recognize diseased cells with downregulated or lacking HLA class I expression, referring to the “missing self” hypothesis 34 . Lacking a specific HLA-I ligand or any responding iKIR, yields hypo-responsive NK clones with high activation threshold 30 . It is particularly noteworthy that  KIR -gene complex diversity influences surface expression, ligand specificities, ligand binding affinity, and subsequent signal transduction through KIR-HLA class I interaction 36 – 38 . Such an intense heterogeneity in  KIR  and  HLA  complex prominently affects NK cell responses and is associated with disease susceptibility 39 . Genome-wide association studies (GWAS) have identified 45 loci associated with lung cancer risk 40 . More specifically, SNPs from 5p15.33, 6p21.33, and 15q25.1 regions are strongly associated with lung cancer in Caucasians 41 . However, the composition of multiple highly-homogeneous gene content, intense polymorphism, strong linkage disequilibrium between multiple loci, and low/no coverage by GWAS reagents pose challenges to studying the polymorphism of KIR (19q13) and HLA (6p21) gene families by GWAS.\nThe present study aimed to investigate the contribution of  KIR  complex and  HLA-I  ligands to the genetic predisposition to lung cancer in the native population of Fars province, located in the southern part of Iran, and to disclose possible associations with the dysfunctional state of NK cells in the context of lung cancer (Table  1 ). Table 1 Characteristics of the study population. Characteristics Lung cancer patients (n = 232) Healthy controls (n = 448) Mean age ± SD 64.4 ± 11.1 58.13 ± 12.66 Gender  Female 34 (14.7%) 150 (33.5%)  Male 198 (85.3%) 298 (66.5%) Smoking status  Smoker 51 (21.98%) 64 (14.3%)  Non-smoker 0 (0.0%) 3 (0.7%)  Unknown 181 (78.02%) 381 (85.0%) Lung cancer subtypes  SCLC 53 (22.8%)  NSCLC 179 (77.2%)   Squamous cell carcinoma 144 (62.1%)   Adenocarcinoma 35 (15.1%)\nCharacteristics of the study population.\n\nThe distribution of 16 KIR genes were determined in patients and HCs (Table  2 ). As observed, framework genes ( 3DL3 ,  3DP1 ,  2DL4 ,  3DL2 ) were presented in all subjects. Two adjacent B haplotype-associated genes  2DL2  (67.2% vs. 55.4%,  p  = 0.003, OR 1.63, CI 1.17–2.26) and  2DS2  (62.9% vs. 54.6%,  p  = 0.041, OR 1.42, CI 1.02–1.96) were significantly increased in patients in comparison with controls. Activating genes  2DS1  (50.2% vs. 41.4%,  p  = 0.029, OR 0.69, CI 0.5–0.95) and  3DS1  (47.3% vs. 37.5%,  p  = 0.015, OR 0.67, CI 0.48–0.92) were more frequently presented in controls than patients, conferring protection against the lung cancer. Table  2  also shows the results with further assessment of  KIR  genes and their associations with lung cancer subtypes (NSCLC, SCLC). Table 2 KIR gene frequencies among patients with lung cancer and healthy controls. KIR  genes Healthy controls Lung cancer Comparisons Lung cancer NSCLC SCLC Lung cancer versus HC NSCLC versus HC SCLC versus HC n = 448 n = 232 n = 179 n = 53 p  value OR (95% CI) p  value OR (95% CI) p  value OR (95% CI) %F (N + /n) %F (N + /n) %F (N + /n) %F (N + /n) Group-A haplotype-associated  KIR  genes 2DL1 98.0 (439/448) 97.4 (226/232) 97.2 (174/179) 98.1 (52/53) 2DL3 88.2 (395/448) 85.3 (198/232) 84.4 (151/179) 88.6 (47/53) 3DL1 92.6 (415/448) 95.3 (221/232) 96.6 (173/179) 92.4 (49/53) 2DS4 92.9 (416/448) 94.8 (220/232) 95.5 (171/179) 92.4 (49/53) 2DS4fl 12.9 (31/242) 10.4 (18/174) 10.2 (14/137) 10.8 (4/37) 2DS4del 73.5 (178/242) 70.6 (123/174) 70.8 (97/137) 70.3 (26/37) 2DS4fl,del 13.6 (33/242) 19.0 (33/174) 19.0 (26/137) 18.9 (7/37) Group-B haplotype-associated  KIR  genes 2DL2 55.4 (248/448) 67.2 (156/232) 65.9 (118/179) 71.6 (38/53) 0.003 1.63 (1.17–2.26) 0.016 1.56 (1.087–2.24) 0.027 2.04 (1.09–3.82) 2DL5 66.1 (296/448) 63.8 (148/232) 64.2 (115/179) 62.2 (33/53) 3DS1 47.3 (212/448) 37.5 (87/232) 36.8 (66/179) 39.6 (21/53) 0.015 0.67 (0.48–0.92) 0.02 0.65 (0.43–0.93) 2DS1 50.2 (225/448) 41.4 (96/232) 41.3 (74/179) 41.5 (22/53) 0.029 0.69 (0.5–0.95) 2DS2 54.5 (244/448) 62.9 (146/232) 62.0 (111/179) 66.0 (35/53) 0.041 1.42 (1.02–1.96) 2DS3 41.5 (186/448) 40.1 (93/232) 39.6 (71/179) 41.5 (22/53) 2DS5 39.5 (177/448) 32.8 (76/232) 32.9 (59/179) 32.0 (17/53) Framework genes/pseudogenes 2DL4 100 (448) 100 (232) 100 (179) 100 (53) 3DL2 100 (448) 100 (232) 100 (179) 100 (53) 3DL3 100 (448) 100 (232) 100 (179) 100 (53) 2DP1 97.8 (438) 96.1 (223) 94.9 (170) 100 (53) 3DP1 100 (448) 100 (232) 100 (179) 100 (53) N + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test.\nKIR gene frequencies among patients with lung cancer and healthy controls.\nN + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test.\nTo evaluate the contribution of  KIR - HLA  combinations to lung cancer risk, we analyzed the distribution of  KIR  genes and cognate  HLA-I  ligands within a group of 85 patients along with 178 HCs (Table  3 ). Similar to the results with individual  KIR  genes, coexistence of  2DL2 / C1  (55.3% vs. 38.3%,  p  = 0.026, OR 1.99, CI 1.11–3.56) and  2DS2 / C1  (50.8% vs. 34.7%,  p  = 0.036, OR 1.93, CI 1.08–3.46) were found to occur more frequently in lung cancer patients than controls. A significantly less frequent carriage of  3DL1 - Bw4  combination was detected in patients with lung cancer than HCs (44.2% vs. 62%,  p  = 0. 014, OR 0.48, CI 0.25–0.91). However, no significant differences were observed between the two groups for the prevalence of  2DS1  and  3DS1  genes combined with respective  HLA-C2  and  HLA-Bw4  ligands, suggesting that particular educational states of NK cells may alter NK cell functionality in the lung cancer setting. Table 3 Frequency of KIR-HLA combinations, and HLA class-I ligands among patients with lung cancer and healthy controls. KIR/HLA Healthy controls Lung cancer Lung cancer versus HC n = 448 n = 232 p  value OR (95% CI) %F (N + /n) F% (N + /n) KIR-binding motif HLA-C1 74.2 (124/167) 81.5 (53/65) HLA-C2 73.6 (123/167) 72.3 (47.65) HLA-Bw4 62.1 (95/153) 48.1 (25/52) Bw4T80 45.7 (70/153) 44.2 (23/52) Bw4I80 16.3 (25/153) 3.8 (2/52) 0.018 0.2 (0.047–0.9) HLA-A3/A11 39.2 (31/79) 38.1 (24/63) HLA-A23/24/25/32 49.3 (39/79) 44.4 (28/63) KIR-HLA combination 3DL2 + A3/A11 39.2 (31/79) 38.1 (24/63) 2DL1 + C2 71.8 (120/167) 72.3 (47/65) 2DL3 + C1 68.8 (115/167) 69.2 (45/65) 2DL2 + C1 38.3 (64/167) 55.3 (36/65) 0.026 1.99 (1.11–3.56) 3DL1 + Bw4 62.0 (95/153) 44.2 (23/52) 0.014 0.48 (0.25–0.91) 2DS1 + C2 26.6 (45/169) 27.7 (18/65) 2DS2 + C1 34.7 (58/167) 50.8 (33/65) 0.036 1.93 (1.08–3.46) 3DS1 + Bw4 20.2 (31/153) 15.3 (8/52) N + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test.\nFrequency of KIR-HLA combinations, and HLA class-I ligands among patients with lung cancer and healthy controls.\nN + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test.\nWhen  HLA-I  ligands were analyzed separately,  HLA-Bw4  ( Ile80 ) was less prevalent (3.8% vs. 16.3%,  p  = 0.018, OR 0.2, CI 0.47–0.9) in lung cancer patients than in controls (Table  3 ). This points to the possibility that the difference in the distribution of  3DL1 - Bw4  combination could be driven by  Bw4  ( Ile80 ) .  The results of comparing the frequency of remaining  HLA-I  genes didn’t reach the level of statistical significance.\nSubgroup analysis wasn’t accomplished regarding the association of  KIR - HLA  combinations and  HLA-I  ligands with different lung cancer subtypes owing to the inadequate sample size included in the HLA typing method.\nKIR genotype profiles of 232 lung cancer patients and 448 HCs are listed in Table  4 . A set of 65 genotypes differentiated by KIR gene content were detected in a total of 680 study participants from southern Iran. Thirty-three genotypes occurred in both patients and controls, 13 genotypes occurred only in patients, and 19 genotypes occurred only in controls.Genotype ID5 (12.1% vs. 7.4%,  p  = 0.048, OR 1.73, CI 1.015–2.93) was significantly more frequent in patients in comparison with controls. Strikingly, the genotype ID19, which is a rare CxTx genotype carrying  2DL2  in the absence of  2DS2  was associated with a more than fivefold increase in lung cancer risk (2.4% vs. 0.4%,  p  = 0.021, OR 5.92, CI 1.18–29.58) (Fig.  1 ). Table 4 KIR genotype frequencies among patients with lung cancer and healthy controls. KIR Healthy controls Lung cancer Comparisons Lung cancer NSCLC SCLC Lung cancer versus HC NSCLC versus HC SCLC versus HC n = 448 n = 232 n = 179 n = 53 p  value OR (95% CI) p  value OR (95% CI) p  value OR (95% CI) %F (N+) %F (N+) %F (N+) %F (N+) KIR genotypes AA 25.7 (115) 19.4 (45) 19.5 (35) 16.9 (9) Bx 74.3 (333) 80.6 (187) 80.4 (144) 83.1 (44) CxT4 22.7 (102) 17.6 (41) 18.4 (33) 15.0 (8) C4Tx 23.4 (105) 28.4 (66) 29.6 (53) 24.5 (13) C4T4 12.7 (57) 9.5 (22) 8.37 (15) 13.2 (7) CxTx 15.4 (69) 25.0 (58) 24.0 (43) 30.2 (16) 0.0035 1.83 (1.23–2.71) 0.015 1.74 (1.13–2.66) 0.011 2.37 (1.25–4.5) C4 Linkage groups 36.2 (162) 37.9 (88) 37.9 (68) 37.7 (20) T4 Linkage groups 35.4 (159) 27.1 (63) 26.8 (48) 28.3 (15) 0.03 0.67 (0.47–0.97) 0.0039 0.66 (0.45–0.97) N  + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test. Figure 1 KIR gene content diversity among patients with lung cancer and healthy controls. N+ : number of individuals positive for the gene; n: number of individuals tested for the gene; Gene content of 65 KIR genotypes are displayed by presence/shaded boxes or absence/white boxes of 16 KIR genes. Distribution of genotypes with ID5, and ID19 highlighted by dark boxes were found to be significantly different between lung cancer patients and controls. * p  = 0.048, OR 1.73, 95% CI: (1.015–2.93); ** p  = 0.021, OR 5.92, 95% CI: (1.18– 29.58).\nKIR genotype frequencies among patients with lung cancer and healthy controls.\nN  + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test.\nKIR gene content diversity among patients with lung cancer and healthy controls. N+ : number of individuals positive for the gene; n: number of individuals tested for the gene; Gene content of 65 KIR genotypes are displayed by presence/shaded boxes or absence/white boxes of 16 KIR genes. Distribution of genotypes with ID5, and ID19 highlighted by dark boxes were found to be significantly different between lung cancer patients and controls. * p  = 0.048, OR 1.73, 95% CI: (1.015–2.93); ** p  = 0.021, OR 5.92, 95% CI: (1.18– 29.58).\nThe distribution of main genotypes AA and Bx were comparable among the two groups. Significant differences in the frequencies of Bx genotype subsets categorized based on C4 and T4 gene clusters were observed, whereby T4 gene cluster (35.4% vs. 27.1%,  p  = 0.03, OR 0.67, CI 0.47–0.97) was found to be associated with reduced risk of lung cancer in our study population. In contrast, we noticed significantly more frequent CxTx subset (25% vs. 15.4%,  p  = 0.0035, OR 1.83, CI 1.23–2.71) in patients indicating an association between this subset and increased risk of lung cancer (Table  4 ). KIR genotype frequencies and their statistical associations with lung cancer subtypes (NSCLC, SCLC) are summarized in Table  4 .\nTo explore further the possible associations of carrying gene contents varied in the number of inhibitory and activating  KIR  genes on susceptibility to lung cancer, we assessed comparisons with regard to different numbers of  iKIRs  and  aKIRs  (Table  5 ). Carriage of genotypes with  iKIRs  >  4  was more likely presented in patients (75% vs. 67%,  p  = 0.035,  P c  = 0.14, OR 1.48, CI 1.036–2.11), on the contrary genotypes with  aKIRs  > 4 were more frequently found in HCs (50% vs. 40.1%,  p  = 0.015,  P c  = 0.06, OR 0.66, CI 0.48–0.92). Lung cancer patients were more likely to carry genotypes with  iKIRs  >  aKIRs  (64.7% vs. 52.2%,  p  = 0.002,  P c  = 0.008, OR 1.67, CI 1.2–2.32), and this difference remained significant after being corrected for multiple comparisons, suggesting the strong association of genotypes with  iKIRs  >  aKIRs  with susceptibility to lung cancer. Table 5 Carrier frequency of various susceptibility-related factor combinations among patients with lung cancer and healthy controls. KIR combination Healthy controls Lung cancer Lung cancer versus HC n = 448 n = 232 p  value Pc OR (95% CI) %F (N + /n) F% (N + /n) KIR genotypes iKIR  >  aKIR 52.2 (234/448) 64.7 (150/232) 0.002 0.008* 1.67 (1.2–2.32) aKIR  >  iKIR 19.4 (87/448) 13.4 (31/232) iKIR  > 4 67.0 (300/448) 75.0 (174/232) 0.035 0.14* 1.48 (1.036–2.11) aKIR  > 4 50.0 (224/448) 40.1 (93/232) 0.015 0.06* 0.66 (0.48–0.92) KIR combined genotypes CxTx/ 2DS2 11.1 (50/448) 20.2 (47/232) 0.0074 0.066** 1.84 (1.18–2.88) CxTx/ 2DL2 12.3 (55/448) 23.7 (55/232) 0.0012 0.011** 2.04 (1.34–3.13) iKIR  >  aKIR  carriers subgroup 2DS2  presence 45.2 (105/232) 59.3 (89/150) 0.0087 0.078** 1.76 (1.16–2.67) 2DL2  presence 49.1 (114/232) 66.6 (100/150) 0.0008 0.0072** 2.05 (1.35–3.17) N + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test;  Pc : corrected  p  values,  Pc* : correction factor = 4,  Pc** : correction factor = 9.\nCarrier frequency of various susceptibility-related factor combinations among patients with lung cancer and healthy controls.\nN + number of individuals positive for the gene,  n  number of individuals tested for the gene,  HC  healthy control,  OR  odds ratio,  CI  confidence interval.  p  < 0.05: statistically significant; based on two-tailed Fisher’s exact test;  Pc : corrected  p  values,  Pc* : correction factor = 4,  Pc** : correction factor = 9.\nWe next performed a comparative analysis to explore whether simultaneous inheritance of disease risk-related factors influences disease susceptibility (Table  5 ). We found that patients with lung cancer were more likely to carry the combination of CxTx/ 2DS2  compared to controls (20.2% vs. 11.1%,  p  = 0.0074,  P c  = 0.066, OR 1.84, CI 1.18–2.88). The likelihood of carrying CxTx/ 2DL2  combination was also significantly higher in patients (23.7% vs. 12.3%,  p  = 0.0012,  P c  = 0.011, OR 2.04, CI 1.34–3.13). Likewise, disease susceptibility was conferred by the presence of  2DL2  within individuals carrying  iKIRs  >  aKIRs  (66.6% vs. 49.1%,  p  = 0.0008,  P c  = 0.0072, OR 2.05, CI 1.35–3.17), this association was weakened in the presence of  2DS2  (59.3% vs. 45.2%,  p  = 0.0087,  P c  = 0.078, OR 1.76, CI 1.16–2.67).\n\nIn the present study, we assessed the contribution of  KIR  gene content and their corresponding  HLA-I  ligands to lung cancer development in the ethnically homogeneous population of southern Iran. Although previous studies have examined KIRs at genetic, transcriptional, and expression levels in lung cancer, to our knowledge, this is the first report that demonstrates individual  KIR  genes and certain genotypes seem to be associated with susceptibility to lung cancer. Given our sizeable dataset, suggestive interactions between KIR-HLA class I ligands can influence the dynamics of NK cell responses in the lung cancer setting.\nDespite existing research addressing the KIR-HLA pair's role in lung cancer, their findings are less consistent. Most recently, in the Chinese Han population, studies conducted by Li et al. 42  and Yu et al. 43 , found no association between  KIRs  and  KIR - HLA  combinations with metastatic NSCLC (mSCLC) and adenocarcinoma, respectively. Consisting with these findings, Wisniewski et al. couldn’t find a significant difference between  KIR  genes or combinations of  KIR - HLA  in 269 Polish Caucasians with NSCLC compared with 690 HCs 44 . However, Wisniewski et al. reported carriers of homozygous  HLA - C1  and  C2  were more frequent in NSCLC patients, which was not detected in our study 44 . Furthermore, Al Omar et al. observed significantly increased  2DL1 / C2  and decreased  2DL3 / C1  in NSCLC patients from England and Northern Ireland 45 . Decreased frequency of  Ile80  allele in NSCLC patients positive for  3DL1 / Bw4  and decreased  Thr80  allele in SCLC patients positive for  3DS1 / Bw4,  which is observed by Al Omar et al. 45 , Conforms to our findings of less frequent  3DS1 ,  3DL1 / Bw4  in lung cancer patients. In part, these inconsistent results may be elucidated by the small sample size, heterogeneity of the target population (study population), and divergent distribution of  KIRs  and  HLAs  in different ethnic groups. Importantly, cross-talk of NK cells and the unique microenvironment of each lung cancer subtype 46  could be responsible for behavioral differences in NK cells, assessing distinct histologic subtypes including (mNSCLC, NSCLC, and SCLC) can presumably lead to conflicting results observed in mentioned studies.\nAlthough we didn’t examine survival rate and response to treatment in lung cancer patients, previous studies obtained interesting results. Yu et al. noted that chemotherapy-treated mNSCLC patients with  KIR2DS4del  and  HLA-Bw4  ( Thr80 ) gene expression at the mRNA level exhibited poor overall survival (OS) 43 . Wisniewski et al. reported the striking association of  2DL2 / 2DS2 / C1  combination with more prolonged survival and better response to therapy in Polish patients 44 , which is discordant with the predisposing effect of  2DL2 / C1 ,  2DS2 / C2  on lung cancer risk observed in our study. Given the cancer setting, it is crucial to consider the impact of chemotherapy agents on the sensitization of tumor cells to NK cell activity. As it has been shown that stress signals induced by chemotherapy and other treatment modalities can elevate the expression of NK cell-activating ligands 47 – 49  or downregulate inhibitory ligands 50 , 51 , the transient deleterious effect of chemotherapeutic agents on NK cells has also been observed 52 , 53 .\nOur findings primarily determined an association between the carriage of  KIR2DL2  and its activating counterpart  2DS2  with an increased risk of lung cancer. We identified similar results when further analyzing  2DL2  and  2DS2  in the presence of their corresponding  HLA-C1  allele. Consistent with our results,  2DL2  has been demonstrated to confer susceptibility to endometriosis 54 , leukemia 55 , and could be predisposing to lymph node metastasis (LNM) in HNSCC as well 56 . The carrier frequency of  2DL2 / C1  in malignant melanoma patients with the advanced stage was significantly higher compared with lower-stage patients 57 . Similar results were reported by Naumova et al. showing the association of  2DL2 / C1  with malignant melanoma 58 . Additionally,  2DL2  and  2DS2  have been shown to confer a predisposition to lymphatic invasion in ER + and PR + breast cancer cases 59 .\nAccording to the “licensing” model, NK cell education via inhibitory receptors integrating with cognate HLA-I ligands endows NK cells with full effector functions and self-tolerance 34 , while NK cell licensing by iKIRs translates into effective sensing of missing HLA I targets, “missing-self” hypothesis, aKIR mediated licensing in the presence of its HLA I ligand induces hypo-responsiveness and renders NK cells impaired responsiveness 60 . Analysis of infiltration pattern and immune cell localization in NSCLC patients revealed that HLA-I negative tumors are predominantly TIL-free and encapsulated by stromal tissue, which consists of a dense structure of FAP + fibroblasts 61 . In addition, reduced TIL infiltration, bigger tumor size, and lymphatic spread have been observed among HLA-I − /PD-L1 +  tumors 62 . To this extent, our finding implies that NK cells of  2DL2 / C1  carriers are incapable of mounting an efficient response against lung cancer tumors sustaining HLA-I expression due to a defect in “missing-self” recognition. Stromal tissue surrounding tumor lesions, which restrains TILs, including NK cells 61 , could hypothetically represent another immune escape mechanism to avoid NK cell attack in lung cancer with total loss or downregulated HLA-I. It is shown that KIRs recognize altered peptides presented by cognate HLA-I ligands 63 , indicating that alterations in peptide repertoire mostly occurring in the process of tumorigenesis could be detected by KIRs 64 . As a result, stimulation of aKIRs with neoantigens and tumor-inducible ligands expressed on lung cancer cells may prompt cytokine release instead of cytolytic function. Supporting examples would be recent studies in which β 2 -microglobulin–independent ligand has been suggested to be recognized by 2DS2 65  and 2DS4 interacting with melanoma-derived non-class I MHC proteins 66 . The 2DS2-mediated education in the presence of C1 ligand could raise activation threshold and cause hypo-responsiveness in carriers of  2DS2 / C1  combination, it also remains possible that upregulated “induced self” ligands mentioned above are unable to overcome such hypo-responsiveness. Due to the tight LD between  2DS2 / 2DL2 , the co-carriage of this combination may exacerbate the detrimental impact attributed to individual genes. More investigation is needed to distinguish the predisposing effect of these two genes on lung cancer.\nMoreover, the frequency of  KIR2DS1  and  3DS1  in the HC group was higher than in patients. We couldn’t detect a significant association of  2DS1/C2 ,  3DS1/Bw4  combinations with lung cancer, though the  HLA-Bw4 (Ile80)  allele and  KIR3DL1 / Bw4  were strongly associated with protection against lung cancer. NK cells expressing 2DS1 exhibit an anergy state in individuals carrying  HLA-C2 / C2 , but not in  HLA-C1 / Cx  carriers 67 . Likewise, Bw4 (Ile80) could be recognized by 3DS1 positive NK cells derived from donors lacking  Bw4  ( Ile80 ), in contrast with NK cells from donors positive for  Bw4  ( Ile80 ) 68 . Referring to these studies, it can be suggested that NK cells generated from those  3DS1  and  2DS1  carriers lacking putative  HLA-Bw4  and  HLA - C2  ligands have lower activation threshold and might recognize different ligands associated with lung tumor transformation. Therefore, carrying  3DS1  and  2DS1  in the absence of cognate HLA class I ligands could confer better protection against lung cancer. Supporting this explanation, HLA-F open conformers (OCs) have been reported as high-affinity ligands for 3DS1 69 , and Kiani et al. have shown that 3DS1 positive NK cells could be activated upon ligation with HLA-F in which stimulation with HLA-F results in increased antiviral function in NK cells 70 . Interestingly a highly expressed level of HLA-F has been detected in lung cancer 71 , suggesting that HLA-F might be a factor in the association of 3DS1/2DS1 with protection in lung cancer. Furthermore KIRs exhibit various degrees of peptide selectivity, implying that KIRs are sensitive to altered peptides, and this sensory mechanism is more sensitive than “missing self-detection” of lowered HLA-I expression on target cells 72 . Regarding iKIRs, several studies have reported changing peptide repertoire in tumor cells function as peptide antagonism, which down-modulates NK cell inhibition by reducing inhibitory ligands on tumor cells, caused by low-affinity interaction of KIR-HLA 73 , 74 . The protective effect conferred by  3DL1 / Bw4  could result from 3DL1 sensitivity to subtle alterations in presented peptides leading to a reduction in inhibitory signals and triggering cytotoxicity in the absence of HLA downregulation.\nEstimating the immunological genetic profile could preferably characterize the pathogenesis of lung cancer. Our findings with more frequent T4 gene cluster carriers in HCs, which is likely imparted by the presence of  3DS1 / 2DS1  combination in T4 gene cluster, are in contravention of CxT4 predisposing role in head and neck squamous cell carcinoma and colorectal adenocarcinoma reported in our previous studies of the same population 75 , 76 . The association of CxTx with lung cancer risk is in line with susceptibility to meningioma reported in CxTx carriers 77 . The results of comparing the different number of  iKIR  and  aKIR  genes, in addition to signifying the strong positive association with lung cancer risk in individuals carrying more inhibitory genes; displayed the influence of activating genes on protection against disease regardless of not being significant after the  p  value correction.\nAs we emphasized the tight LD between  2DL2  and  2DS2 , it is difficult to dissect the contribution of individual  2DL2  and  2DS2  genes in susceptibility to the disease. Hence, assessing rare genotypes lacking either  2DL2  or  2DS2  is highly informative. Accordingly, we noticed a striking association of the genotype ID19, a rare CxTx genotype carrying  2DL2  in the absence of  2DS2,  with increased lung cancer risk by more than fivefold, suggesting a predominant detrimental impact of  2DL2  over  2DS2 . Regarding our analysis of co-existing susceptibility factors with lung cancer, simultaneous inheritance of CxTx/ 2DL2  was shown to predispose carriers to lung cancer. Although, the simultaneous presence of CxTx/ 2DS2  did not meet the significant level after  p  value correction and seems to confer a slightly lower risk of lung cancer compared to CxTx/ 2DL2 . Superior adverse effect was noticeably related to individuals with  iKIR  >  aKIR  in the presence of  2DL2  rather than  2DS2 . Our findings are strengthened by the report denoting the correlation of higher expression in inhibitory  KIRs  with poor prognosis in lung cancer patients. A higher proportion of NK cells expressing inhibitory KIRs was noticed in NSCLC patients in which lower cytotoxicity and reduced IFN - γ production were also shown 78 . Consistently, separate consideration of inhibitory and activating counterparts demonstrated the susceptibility of  2DL1 + / S1 −  genotype carriers to cutaneous melanoma and the formation of sentinel lymph node metastasis within individuals with homozygous  HLA - C2 79 . A previous study by Momot et al. suggested that carrying  2DS2 + / L2 −  combination is related to a higher risk of scleroderma disease 80 , and similar results were illustrated regarding systemic sclerosis 81 . However, this disagreement with our observations might be due to different mechanisms involved in tumorigenesis and autoimmune disorders. The fact that the difference of genotypes with  iKIRs  >  aKIRs  and CxTx combined with  2DL2  but not  2DS2  remained significant after correction of  p  values implies that these combinations are significantly more strongly associated with lung cancer development. It can also be presumed that the presence of  2DL2  intensifies disease associations.\nIt is important to highlight the suppressive effects on NK cells suggested to be driven by alveolar macrophages and epithelial lining fluid of the lower respiratory tract 82 . Despite comprising a well-differentiated phenotype, NK cells in lung tissue are exposed to a restricting microenvironment in homeostasis, causing a hypo-functional state of lung NK cells to stimuli in comparison with NK cells of peripheral blood 83 , 84 . Attenuated killing potency in TME owing to the presence of regulatory T cells (TREGs) and myeloid-derived suppressor cells (MDSCs), limitations of leukocyte infiltration, as well as immunosuppressive factors in lung TME such as adenosine and transforming growth factor β (TGF-β) are yet to be overcome 4 , 85 . Taken together, it can be speculated that carrying more inhibitory gene content may interfere with NK-mediated immunosurveillance, favoring tumor evasion in the existing suppressor context of lung tissue. On the other end, activating gene content is presumed to restore the functional competence of NK cells, especially in suppressive lung settings.\nIn conclusion, we report new susceptibility factors and the contribution of  KIR  and  HLA - I  encoding genes to lung cancer risk, highlighting an array of genetic effects and disease setting that regulates NK cell responsiveness. Our results suggest that inherited  KIR  genes and  HLA-I  ligands specifying the educational state of NK cells can modify lung cancer risk. In the current study,  HLA - I  ligands and co-associations of  KIR - HLA  were examined for a limited number of subjects; also an inadequate number of patients with different lung cancer subtypes hindered the evaluation of  KIRs  and  HLA-I  genes impact within subgroups. Thus, larger cohorts assessing the contribution of  KIR - HLA  combinations are needed to confirm these associations. Functional analysis might help extend associations to the potential therapeutic strategies against lung cancer. The unique microenvironment of each lung cancer subtype with a varied composition of immune cells is assumed to affect NK cell characteristics 86 . Further investigations on mechanisms involved in NK cell dysfunction in different subtypes might develop into NK-based immunotherapies in lung cancer.\n\nA total of 232 patients with lung cancer (comprising NSCLC: squamous cell carcinoma and adenocarcinoma, SCLC subtypes) and 448 healthy controls (HC) from a homogenous population of the southern part of Iran (Fras province) were included in this case–control study. The enrolled group of 232 unrelated lung cancer patients was made up 85.3% of men and 14.7% of women with a mean age of 64.4 ± 11.1. Healthy controls were comprised 66.5% of men and 33.5% of women with a mean age of 58.13 ± 12.66. The pathologically confirmed lung cancer cases were recruited from Faghihi hospital, Shiraz University of medical sciences. Age and sex-matched HCs with no Family History of Cancer (FHC) were selected from the Motahari clinic. The demographic and clinical characteristics of lung cancer patients were gathered from medical records (Table  1 ). Due to the fact that smoking status for a very small portion of our lung cancer patients and HCs was available, stratification of the study population based on smoking status wasn’t carried out.\nInformed consent from all research participants was obtained, and the study was carried out according to the declaration of Helsinki. This study was reviewed and ethically approved by the Medical Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1398.1110).\nGenomic DNA extraction from whole blood samples was performed using QIAamp DNA Mini Kit (Qiagen, Germany) as detailed in the manufacturer’s instructions. The PCR-SSP method was used for genotyping 16  KIR  genes, and  KIR2DS4  variants ( KIR2DS4fl :  2DS4  full variant, and  KIR2DS4del :  2DS4  deleted variant) as previously described 87 . Detailed information on primer sequences, thermal conditions, and the mixture of each reaction are reported in our previous study 59 . Reference DNA samples from the UCLA KIR exchange program provided by Prof. Raja Rajalingam were applied to ensure typing accuracy. An alternative SSP-PCR method was used to confirm the unique and unusual KIR genotyping 88 .\nThe KIR-binding  HLA-A ,  B,  and  C  ligands of 85 lung cancer patients and 178 HCs were typed using the recently developed direct DNA sequencing method. The strategy includes PCR amplifying exons 2 and 3 using  HLA-A ,  B,  or  C  gene-specific primers and direct sequencing of the segment that encodes the KIR-binding region. The process was accomplished in accordance with the method described by Ashouri et al. 89 .\nKIR genotypes of the study participants were assigned according to previous studies 88 , 90 . The genotype AA comprises fixed gene content ( 2DL3-2DL1-2DP1-3DL1-2DS4)  surrounded by frameworks. Carriers of AA genotype-related genes were regarded as homozygous AA, and the remaining subjects were considered as Bx genotype carriers which can be heterozygous AB or homozygous BB. The KIR genotype ID was obtained using the allele frequency database ( http://www.allelefrequencies.net/ ) for all participants.\nBased on the linkage disequilibrium, two frequently occurring clusters that include distinct sets of B-haplotype-specific KIR genes have been identified 91 . C4 linkage group comprising  KIR2DS2-2DL2-2DS3-2DL5B  genes is located in the centromeric region of the KIR complex, while the T4 linkage group contains  KIR3DS1 - 2DL5A - 2DS5 - 2DS1  genes located at the telomeric region of the complex. Concerning the presence or absence of C4 and T4 linkage groups, The Bx genotype carriers were further divided into the following four subsets: C4Tx, CxT4, C4T4, and CxTx 89 . The frequency of C4 and T4 gene clusters were defined by subsequent formulas: C4 = nC4Tx + nC4T4, and T4 = nCxT4 + nC4T4 (n: number of individuals with a particular subset within each group).\nThe percentage of  KIR  genes in both study groups was indicated by direct counting (number of subjects positive for the gene divided by the number of subjects per population × 100). Differences in the distribution of each  KIR  gene, genotypes, KIR-binding HLA ligands, and KIR-HLA pairs between lung cancer patients and HCs were estimated by two-tailed Fisher Exact probability ( p ) test using SPSS (IBM, US) version 16.0 and Items with  p  < 0.05 were considered as statistically significant. Moreover, the Odds ratio (OR) and 95% Confidence Intervals (CI) were calculated to assess the magnitude of associations. The method expounded by Svejgaard and Ryde 92  was applied to identify the combined effect of the lung cancer susceptibility factors CxTx + /2DL2 +  and CxTx + /2DS2 + .  p  values regarding the association of genotypes with certain number of genes ( aKIRs  >  iKIRs ,  iKIRs  >  aKIRs ,  aKIRs  > 4,  iKIRs  > 4) were corrected using  P n  = 1 − (1 −  P ) n , where n represents the number of comparisons 92 .\nEthical approval of the research was confirmed by the Medical Ethics Committee of Shiraz University of Medical Sciences [IR. SUMS.REC.1398.1110].","source_license":"CC-BY-4.0","license_restricted":false}