Up-regulation of activating and inhibitory NKG2 receptors in allogeneic and autologous hematopoietic stem cell grafts

Background

Hematopoietic Stem Cell Transplantation (HSCT) is known to induce the inhibitory immune receptor NKG2A on NK cells of donor origin. This occurs in allogeneic recipients, in both the haploidentical and HLA-matched settings.

Methods

To gain further insight, not only NKG2A, but also the activating receptors NKG2C and NKG2D were assessed by flow cytometry. Immunophenotyping was carried out not only on CD56⁺ but also on CD8⁺ lymphocytes from leukemia and lymphoma patients, receiving both HLA-matched (n = 7) and autologous (n = 5) HSCT grafts. Moreover, cognate NKG2 ligands (HLA-E, MICA, ULBP-1, ULBP-2 and ULBP-3) were assessed by immunohistochemistry in diagnostic biopsies from three autotransplanted patients, and at relapse in one case.

Results

All the NKG2 receptors were simultaneously up-regulated in all the allotransplanted patients on CD8⁺ and/or CD56⁺ cells between 30 and 90 days post-transplant, coinciding with, or following, allogeneic engraftment. Up-regulation was of lesser entity and restricted to CD8⁺ cells in the autotransplantation setting. The phenotypic expression ratio between activating and inhibitory NKG2 receptors was remarkably similar in all the patients, except two outliers (a long survivor and a short survivor) who surprisingly displayed a similar NKG2 activation immunophenotype. Tumor expression of 2 to 3 out of the 5 tested NKG2 ligands was observed in 3/3 diagnostic biopsies, and 3 ligands were up-regulated post-transplant in a patient.

Conclusions

Altogether, these results are consistent with a dual (activation-inhibition) NK cell re-education mode, an innate-like T cell re-tuning, and a ligand:receptor interplay between the tumor and the immune system following HSCT including, most interestingly, the up-regulation of several activating NKG2 ligands. Turning the immune receptor balance toward activation on both T and NK cells of donor origin may complement ex vivo NK cell expansion/activation strategies in unmanipulated patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0213-y) contains supplementary material, which is available to authorized users.     

HLA-E siRNA沉默肝癌细胞HLA-E基因的表达

目的针对HLA-EmRNA靶序列不同位点,设计合成多个siRNA链,定量分析其对HLA-E(+)肝癌BEL-7402细胞的基因沉默效率,筛选最佳抑制效果的siRNA。方法用IFN-γ(5×105IU·L-1)诱导BEL-7402细胞表达HLA-E基因,经流式细胞技术纯化后作为靶细胞。设计并合成3条HLA-E siRNA链(A、B、C),将各siRNA(0.1mmol·L-1)经脂质体Lipofectamin 2000转染至靶细胞。采用细胞免疫荧光、流式细胞技术、Western杂交、实时PCR等定量方法,比较48h后各siRNA的基因沉默效果,并观察HLA-E基因沉默对NK肿瘤细胞杀伤的影响。结果与空白对照、非特异组相比,3组(A、B、C组)HLA-E siRNA均明显抑制细胞HLA-E抗原、蛋白产物、mRNA、细胞表面HLA-E分子的表达(P<0.01),B、C组抑制效果接近90%,高于A组(P<0.01)。A、B、C组NK肿瘤杀伤率升高(P<0.01),而B、C组肿瘤杀伤效果高于A组(P<0.01)。结论经筛选的HLA-EsiRNA能特异、高效沉默肝癌细胞HLA-E基因表达,可能抑制其非经典HLA-Ⅰ途径免疫逃避,为肝癌"基因-免疫"治疗提供新的治疗策略。     

Combined analysis of HLA class I,HLA-E and HLA-G predicts prognosis in colon cancer patients

Background:

Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumour development in colon cancer. The goal of this study was to establish a tumour profile based on biomarkers that reflect a tumour''s immune susceptibility status and to determine their relation to patient outcome.

Methods:

The study population consisted of 285 stage I-IV colon cancer patients of which a tissue micro array (TMA) was available. Sections were immunohistochemically stained for the presence of Foxp3+ cells and tumour expression of HLA Class I (HLA-A, -B, -C) and non-classical HLA-E and HLA-G. All markers were combined for further analyses, resulting in three tumour immune phenotypes: strong immune system tumour recognition, intermediate immune system tumour recognition and poor immune system tumour recognition.

Results:

Loss of HLA class I expression was significantly related to a better OS (P-value 0.005) and DFS (P-value 0.008). Patients with tumours who showed neither HLA class I nor HLA-E or -G expression (phenotype a) had a significant better OS and DFS (P-value <0.001 and 0.001, respectively) compared with phenotype b (OS HR: 4.7, 95% CI: 1.2–19.0, P=0.001) or c (OS HR: 8.2, 95% CI: 2.0–34.2, P=0.0001). Further, the tumour immune phenotype was an independent predictor for OS and DFS (P-value 0.009 and 0.013, respectively).

Conclusion:

Tumours showing absence of HLA class I, HLA-E and HLA-G expressions were related to a better OS and DFS. By combining the expression status of several immune-related biomarkers, three tumour immune phenotypes were created that related to patient outcome. These immune phenotypes represented significant, independent, clinical prognostic profiles in colon cancer.     

HLA—E cDNA克隆及在LcL721.221细胞的表达

目的 克隆HLA-E cDNA,并使其在HLA I类阴性的靶细胞LcL721.221细胞上获得稳定表达。方法 用RT-PCR方法从人外周血淋巴细胞扩增出HLA-E cDNA,并通过内核糖体进入位点（IRES）将目的基因亚克隆于已经载有HLA-A2的逆转录病毒表达载体pGCEN上,构建成HLA-A2/E 多顺反子表达载体（pG/A2E）,采用感染的方法将重组质粒转入LcL721.221细胞,最后经G418筛选及有限稀释,利用抗HLA-E特异的单克隆抗体3D12进行FACS检测,以观察HLA-E分子在靶细胞表面的表达情况。结果 HLA-E 分子在经pG/A2E转染的靶细胞表面获得明显的表达（88.79%）,且显著高于表达HLA-E的对照细胞株JAR（26.21%）,而经pG/A2载体转染的靶细胞则未获得表达。结论 成功构建了pG/A2E多顺反了表达载体,并使HLA-E分子在HLA I类阴性的LcL721.221细胞表面获得表达。     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

The prognostic benefit of tumour-infiltrating Natural Killer cells in endometrial cancer is dependent on concurrent overexpression of Human Leucocyte Antigen-E in the tumour microenvironment

BackgroundHuman Leucocyte Antigen- E (HLA-E) has been reported as both a positive and negative prognostic marker in cancer. This apparent discrepancy may be due to opposing actions of HLA-E on tumour-infiltrating immune cells. Therefore, we evaluated HLA-E expression and survival in relation to the presence of intratumoural natural killer (NK) cells and cytotoxic T cells (CTLs).MethodsTissue microarrays (TMAs) of endometrial tumours were used for immunohistochemical staining of parameters of interest. The combined impact of clinical, pathological and immune parameters on survival was analysed using log rank testing and Cox regression analyses.ResultsUpregulation of HLA-E was associated with an improved disease-free and disease-specific survival in univariate analysis (HR 0.58 95% CI 0.37–0.89; HR 0.42 95% CI 0.25–0.73, respectively). In multivariate analysis, the presence of NK cells predicts survival with a hazard ratio (HR) 0.28 (95% confidence interval (CI) 0.09–0.91) when HLA-E expression is upregulated; but it is associated with a worse prognosis when HLA-E expression is normal (HR 13.43, 95% CI 1.70–106.14). By contrast, the prognostic benefit of T cells was not modulated by HLA-E expression.ConclusionsTaken together, we demonstrate that the prognostic benefit of NK cells, but not T-cells, is influenced by HLA-E expression in endometrial cancer (EC) and propose a model to explain our observations.     

Nonclassical human leukocyte antigen (HLA-G,HLA-E,and HLA-F) in coronary artery disease

AimsSeveral evidences suggest the association between the evolution of coronary artery disease (CAD) and the development of coronary syndrome that is often associated with disrupted plaque and partial or complete thrombosis of the related artery. Because of the inflammatory nature of CAD, we investigated the human leukocyte antigen (HLA)-G, HLA-E, and HLA-F genetic polymorphisms within CAD patients and evaluated their potential association with this disease in Tunisian population.MethodsDifferent polymorphisms in HLA-G (14-bp Insertion/Deletion, +3142C/G), HLA-E (HLA-E*01:01/01:03 A/G), HLA-F (HLA-F*01:02 T/C, 01:03 C/T, 01:04 A/C) genes were typed using different laboratory techniques in a cohort of 89 CAD patients and 84 controls.ResultsA significant association was reported between the HLA-G +3142 G allele (OR = 1.64, 95% CI = 1.05–2.56, p = 0.02) and increased risk of CAD. No association was found for the other studied polymorphisms. When we considered the haplotypes, we found TDELCA and TDELGG haplotypes associated to CAD with p = 0.008 and p = 0.030, respectively, suggesting the potential interaction between HLA-G and HLA-E genes.ConclusionsOur findings indicated that the HLA-G +3142C/G polymorphism and TDELCA and TDELGG haplotypes can harbour a reliable diagnosis value for the risk of CAD development suggesting that HLA-G, -E and -F molecules might be involved in the pathogenesis of the disease. However, further studies are necessary to confirm our results.     

HLA-E基因与allo-HSCT移植效果关系的研究

目的： 探讨HLA-E基因与异基因造血干细胞移植（allo-HSCT）效果的关系。方法：用PCR-SSP方法分别检测22对allo-HSCT供者及受者的HLA-E基因型，并分2组：供受者HLA-E相合组和供受者HLA-E不合组。比较2组在移植后植入率、移植物抗宿主病（GVHD）、免疫重建、自体恢复或恶性病复发等方面的差异。结果：22对移植病例的供受者中，共检出3个HLA-E等位基因，分别为E*0101、E*01031和E*01032，未检出E*0102和*0104。供受者HLA-E相合组9对，供受者HLA-E不合组13对。两组在移植后植入率、GVHD、免疫重建、自体恢复或恶性病复发等无统计学差异。结论：HLA-E基因多态性与allo-HSCT移植效果未见相关性。     

Polymorphisms within the human leucocyte antigen-E gene and their associations with susceptibility to rheumatoid arthritis as well as clinical outcome of anti-tumour necrosis factor therapy

Involvement of the non-classical human leucocyte antigen-E (HLA-E) in both innate and acquired immune response suggests its possible role in development of autoimmune pathologies. This study was undertaken to investigate relationships between the HLA-E gene single nucleotide polymorphisms (SNPs) and a risk of rheumatoid arthritis (RA), as well as to evaluate a potential of these polymorphisms to modulate clinical outcome of anti-tumour necrosis factor (TNF) treatment in female patients. A total of 223 female patients with RA receiving anti-TNF biological therapy and 134 female healthy subjects were enrolled into the study. Genotypings for two SNPs within the HLA-E gene (rs1264457 HLA-E*01:01/01:03; rs1059510 HLA-E*01:03:01/01:03:02) were performed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays. Clinical response was evaluated according to the European League Against Rheumatism (EULAR) criteria at 12 and 24 weeks after initiation of the therapy. The frequency of the HLA-E*01:01/01:01 genotype was decreased significantly in RA patients in comparison to controls (P = 0·031). The presence of the HLA-E*01:01/01:01 genotype in patients correlated with better EULAR response after 12 weeks of anti-TNF treatment, while 01:03 allele carriers were generally unresponsive to the treatment (P = 0·014). The HLA-E*01:03/01:03 genotype was also over-represented among non-responding patients in comparison to HLA-E*01:01/01:01 homozygotes (P = 0·021). With respect to the HLA-E rs1059510 variation, a better response after 12 weeks was observed more frequently in patients carrying the HLA-E*01:03:01/01:03:01 genotype than other genotypes (P = 0·009). The results derived from this study imply that HLA-E polymorphisms may influence RA susceptibility and affect clinical outcome of anti-TNF therapy in female RA patients.