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Human Leukocyte Antigen G Up-Regulation in Lung Cancer Associates with High-Grade Histology, Human Leukocyte Antigen Class I Loss and Interleukin-10 Production

2001; Elsevier BV; Volume: 159; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)61756-7

ISSN

1525-2191

Autores

Mirjana Urosevic, Michael Kurrer, Jivko Kamarashev, Beatrix Mueller, Walter Weder, G. Burg, Rolf A. Stahel, Reinhard Dummer, Andreas Trojan,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

Immune evasion in lung cancer results from both structural and functional alterations of human leukocyte antigen (HLA) class I molecules and the local release of immunosuppressive cytokines. Recent data suggest that HLA-G, a nonclassical class Ib molecule, is involved in immune evasion by tumor cells. We sought to determine whether HLA-G could contribute to immunescape in lung cancer. All of 19 tumor specimens examined demonstrated detectable membrane-bound (HLA-G1), as well as soluble (HLA-G5) isoform transcription. Nine of 34 (26%) tumors were positive by immunohistochemistry using monoclonal antibody (mAb) 4H84, recognizing all denatured HLA-G isoforms, of which six were positive using mAb 16G1, recognizing soluble HLA-G. HLA-G immunoreactivity correlated with high-grade histology, with HLA-G being preferentially expressed on large-cell carcinomas. In these patients, loss of classical HLA class I molecules was observed to associate with HLA-G protein up-regulation. Moreover, we found interleukin-10 expressed in 15 of 34 (44%) tumors, and in most of the HLA-G-positive cases (7 of 9), suggesting up-modulation of HLA-G by interleukin-10. It is conceivable that HLA-G expression in lung cancer might be one of the ways how the tumor down-regulates host immune response, in addition to interleukin-10 production and HLA class I loss. Immune evasion in lung cancer results from both structural and functional alterations of human leukocyte antigen (HLA) class I molecules and the local release of immunosuppressive cytokines. Recent data suggest that HLA-G, a nonclassical class Ib molecule, is involved in immune evasion by tumor cells. We sought to determine whether HLA-G could contribute to immunescape in lung cancer. All of 19 tumor specimens examined demonstrated detectable membrane-bound (HLA-G1), as well as soluble (HLA-G5) isoform transcription. Nine of 34 (26%) tumors were positive by immunohistochemistry using monoclonal antibody (mAb) 4H84, recognizing all denatured HLA-G isoforms, of which six were positive using mAb 16G1, recognizing soluble HLA-G. HLA-G immunoreactivity correlated with high-grade histology, with HLA-G being preferentially expressed on large-cell carcinomas. In these patients, loss of classical HLA class I molecules was observed to associate with HLA-G protein up-regulation. Moreover, we found interleukin-10 expressed in 15 of 34 (44%) tumors, and in most of the HLA-G-positive cases (7 of 9), suggesting up-modulation of HLA-G by interleukin-10. It is conceivable that HLA-G expression in lung cancer might be one of the ways how the tumor down-regulates host immune response, in addition to interleukin-10 production and HLA class I loss. Tumor escape from host immunosurveillance is frequently attributed to the release of immunosuppressive cytokines or the alteration of antigen-presenting molecules on neoplastic cells.1Smith DR Kunkel SL Burdick MD Wilke CA Orringer MB Whyte RI Strieter RM Production of interleukin-10 by human bronchogenic carcinoma.Am J Pathol. 1994; 145: 18-25PubMed Google Scholar, 2Korkolopoulou P Kaklamanis L Pezzella F Harris AL Gatter KC Loss of antigen-presenting molecules (MHC class I and TAP-1) in lung cancer.Br J Cancer. 1996; 73: 148-153Crossref PubMed Scopus (165) Google Scholar, 3Hiraki A Kaneshige T Kiura K Ueoka H Yamane H Tanaka M Harada M Loss of HLA haplotype in lung cancer cell lines: implications for immunosurveillance of altered HLA class I/II phenotypes in lung cancer.Clin Cancer Res. 1999; 5: 933-936PubMed Google Scholar, 4Yanagawa H Takeuchi E Suzuki Y Hanibuchi M Haku T Ohmoto Y Sone S Production of interleukin-10 by alveolar macrophages from lung cancer patients.Respir Med. 1999; 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61: 1138-1149Crossref PubMed Scopus (262) Google Scholar HLA-G is shown to protect HLA class I-deficient targets from NK-mediated lysis, through interactions with killer inhibitory receptors on NK cells.13Rouas Freiss N Khalil-Daher I Riteau B Menier C Paul P Dausset J Carosella ED The immunotolerance role of HLA-G.Semin Cancer Biol. 1999; 9: 3-12Crossref PubMed Scopus (68) Google Scholar, 15Khalil-Daher I Rouas-Freiss N Carosella ED Dausset JB Human leukocyte antigen-G: immunotolerant major histocompatibility complex molecule in transplantation.World J Surg. 2000; 24: 819-822Crossref PubMed Scopus (5) Google Scholar, 16Pazmany L Mandelboim O Vales Gomez M Davis DM Becker TC Reyburn HT Seebach JD Hill JA Strominger JL Human leucocyte antigen-G and its recognition by natural killer cells.J Reprod Immunol. 1999; 43: 127-137Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 17Lopez-Botet M Navarro F Llano M How do NK cells sense the expression of HLA-G class Ib molecules?.Semin Cancer Biol. 1999; 9: 19-26Crossref PubMed Scopus (40) Google Scholar The fact that lymphocytes, macrophages, and dendritic cells harbor HLA-G-specific inhibitory ligands, such as p49, ILT-2, and ILT-4,18Colonna M Navarro F Bellon T Llano M Garcia P Samaridis J Angman L Cella M Lopez Botet M A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells.J Exp Med. 1997; 186: 1809-1818Crossref PubMed Scopus (784) Google Scholar, 19Colonna M Samaridis J Cella M Angman L Allen RL O'Callaghan CA Dunbar R Ogg GS Cerundolo V Rolink A Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules.J Immunol. 1998; 160: 3096-3100PubMed Google Scholar, 20Cantoni C Verdiani S Falco M Pessino A Cilli M Conte R Pende D Ponte M Mikaelsson MS Moretta L Biassoni R p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily.Eur J Immunol. 1998; 28: 1980-1990Crossref PubMed Scopus (151) Google Scholar elucidated HLA-G-mediated inhibition of cytotoxic T cell (CTL)-specific response.21Le Gal FA Riteau B Sedlik C Khalil Daher I Menier C Dausset J Guillet JG Carosella ED Rouas Freiss N HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes.Int Immunol. 1999; 11: 1351-1356Crossref PubMed Scopus (278) Google Scholar To date, transcription of HLA-G has been reported in a variety of human malignancies, including lung cancer.22Real LM Cabrera T Collado A Jimenez P Garcia A Ruiz Cabello F Garrido F Expression of HLA G in human tumors is not a frequent event.Int J Cancer. 1999; 81: 512-518Crossref PubMed Scopus (71) Google Scholar, 23Pangault C Amiot L Caulet Maugendre S Brasseur F Burtin F Guilloux V Drenou B Fauchet R Onno M HLA-G protein expression is not induced during malignant transformation.Tissue Antigens. 1999; 53: 335-346Crossref PubMed Scopus (58) Google Scholar, 24Paul P Cabestre FA Le Gal FA Khalil Daher I Le Danff C Schmid M Mercier S Avril MF Dausset J Guillet JG Carosella ED Heterogeneity of HLA-G gene transcription and protein expression in malignant melanoma biopsies.Cancer Res. 1999; 59: 1954-1960PubMed Google Scholar, 25Paul P Rouas-Freiss N Khalil-Daher I Moreau P Riteau B Le Gal FA Avril MF Dausset J Guillet JG Carosella ED HLA-G expression in melanoma: a way for tumor cells to escape from immunosurveillance.Proc Natl Acad Sci USA. 1998; 95: 4510-4515Crossref PubMed Scopus (387) Google Scholar Although HLA-G protein expression was recently described on intratumoral macrophages and dendritic cells,23Pangault C Amiot L Caulet Maugendre S Brasseur F Burtin F Guilloux V Drenou B Fauchet R Onno M HLA-G protein expression is not induced during malignant transformation.Tissue Antigens. 1999; 53: 335-346Crossref PubMed Scopus (58) Google Scholar, 26Onno M Le Friec G Pangault C Amiot L Guilloux V Drenou B Caulet-Maugendre S Andre P Fauchet R Modulation of HLA-G antigens expression in myelomonocytic cells.Hum Immunol. 2000; 61: 1086-1094Crossref PubMed Scopus (45) Google Scholar the HLA-G up-regulation on lung cancer cells remains controversial.22Real LM Cabrera T Collado A Jimenez P Garcia A Ruiz Cabello F Garrido F Expression of HLA G in human tumors is not a frequent event.Int J Cancer. 1999; 81: 512-518Crossref PubMed Scopus (71) Google Scholar, 23Pangault C Amiot L Caulet Maugendre S Brasseur F Burtin F Guilloux V Drenou B Fauchet R Onno M HLA-G protein expression is not induced during malignant transformation.Tissue Antigens. 1999; 53: 335-346Crossref PubMed Scopus (58) Google ScholarIn lung cancer, the secretion of immunosuppressive Th2 type cytokines, dominated by interleukin-10, is a frequent event and contributes to the progression of disease.1Smith DR Kunkel SL Burdick MD Wilke CA Orringer MB Whyte RI Strieter RM Production of interleukin-10 by human bronchogenic carcinoma.Am J Pathol. 1994; 145: 18-25PubMed Google Scholar, 27Ito N Nakamura H Tanaka Y Ohgi S Lung carcinoma: analysis of T helper type 1 and 2 cells and T cytotoxic type 1 and 2 cells by intracellular cytokine detection with flow cytometry.Cancer. 1999; 85: 2359-2367Crossref PubMed Scopus (95) Google Scholar, 28Sharma S Stolina M Lin Y Gardner B Miller PW Kronenberg M Dubinett SM T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function.J Immunol. 1999; 163: 5020-5028PubMed Google Scholar, 29De Vita F Orditura M Galizia G Romano C Roscigno A Lieto E Catalano G Serum interleukin-10 levels as a prognostic factor in advanced non-small cell lung cancer patients.Chest. 2000; 117: 365-373Crossref PubMed Scopus (127) Google Scholar By down-regulating HLA class I expression and selective HLA-G induction on tumor cells,30Moreau P Adrian Cabestre F Menier C Guiard V Gourand L Dausset J Carosella ED Paul P IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes.Int Immunol. 1999; 11: 803-811Crossref PubMed Scopus (369) Google Scholar interleukin (IL)-10 could contribute to an impaired immune recognition of the tumor.28Sharma S Stolina M Lin Y Gardner B Miller PW Kronenberg M Dubinett SM T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function.J Immunol. 1999; 163: 5020-5028PubMed Google Scholar, 31Matsuda M Salazar F Petersson M Masucci G Hansson J Pisa P Zhang QJ Masucci MG Kiessling R Interleukin 10 pretreatment protects target cells from tumor- and allo-specific cytotoxic T cells and downregulates HLA class I expression.J Exp Med. 1994; 180: 2371-2376Crossref PubMed Scopus (294) Google ScholarUsing real-time quantitative polymerase chain reaction (PCR) (LightCyclerTM) we sought to determine HLA-G transcriptional levels in lung cancer. In addition, we assessed HLA-G protein expression by immunohistochemistry. Here we report that full-length membrane-bound (HLA-G1) and soluble (HLA-G5) message was detectable in all tumor specimens and controls. However, only a group of tumors displayed HLA-G immunoreactivity, of which some were as well positive for expression of the soluble isoform. HLA-G protein expression correlated with both histological tumor type and grade. Loss of classical HLA class I molecules coincided with HLA-G protein up-regulation. All HLA-G-expressing tumors demonstrated IL-10 immunoreactivity.Materials and MethodsTissue Samples and Cell LinesA total of 34 lung cancer samples were collected after surgical resection, after obtaining a previous informed consent of the patient. The patients’ data, histological diagnoses, and assigned stage are summarized in Table 1. Normal lung tissue as internal control was available from seven patients. The human choriocarcinoma HLA-G-positive cell line, JEG-3 (American Type Culture Collection, Manassas, VA) was cultured in Dulbecco's modified Eagle's medium (Biochrom KG, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum with antibiotics-antimycotic (GibcoBRL, Life Technologies AG, Basel, Switzerland), 1 mmol/L sodium-pyruvate (GibcoBRL, Life Technologies AG) and 2 mmol/Ll-glutamine (Biochrom KG). First trimester trophoblast tissue was obtained from elective termination of pregnancy after informed consent of the patient, and used as an additional positive control.Table 1Clinical, Histological, and Immunohistochemical Data from Lung Cancer PatientsNo.SexAgeTumor histologyTumor gradeClinical stageHLA-G protein expressionsHLA-G protein expressionpan-HLA class I expressionIL-10 protein expressionInfiltrating NK cellsT1M53SCCIIIIIbR−FL−−T2M66SCCIIIbR−CL−+T3M78LCCIIIIIIaTTCL++T4M59LCCIIIIbT, R, STCL++T5F62ACIIaR−No loss+−T6M78LCCIIIIbT, R, STCL++T7F76ACIIIa−−CL−+T8F52LCCIIIIIIaT, R, STCL++T9M48SCLC−extensive diseaseT, R, STFL+−T10M69ACIIIIbR−FL++T11M70SCCIIIIbR, S−CL+−T12M63SCCIIIIIbR, S−CL++T13M76SCCIIIIIbR−No loss−−T14F54SCCIIIIbR−FL−−T15M60ACIIIIIbT, RTCL++T16F71ACIIIbR, S−FL−+T17F57LCCIIIIbT, S−CL++T18F56ACIIIIIbT, S−FL−−T19M72LCCIIIIIIb−−CL−+T20M48SCCIIIbT−FL−+T21M67ACIIIb−−CL+−T22M65ACIIbR−FL−−T23M66ACIIIbS−CL−−T24M68ACIIb−−FL−−T25M62ACIIIIAR, S−FL−−T26F74ACIIIa−−FL+−T27M68ACIIIIaS−CL++T28M71ACIIb−−CL−−T29M74SCCIIIaR, S−FL+−T30M61SCCIIIIIaS−FL−+T31M66ACIIIVR−FL−+T32M65ACIIIbR, S−CL−+T33M76ACIIIIbR−CL−−T34M59SCCIIIaR−CL−+M, male; F, female; SCC, squamous cell carcinoma; LCC, large cell carcinoma; AC, adenocarcinoma; SCLC, small cell lung cancer; R, immunoreactivity of residual bronchial structures; S, immunoreactivity of tumor-surrounding lung; T, tumor immunoreactivity; CL, complete loss of pan-class I expression (defined as less than 25% of the cells showing immunoreactivity with TP25.99 mAb); FL, focal loss of pan-class I expression (circumscribed area of TP25.99 immunonegative cells within the tumor); +, immunoreactivity; −, no immunoreactivity with anti-sHLA-G, anti-IL-10 or anti-CD56 mAb. Open table in a new tab RNA Extraction and cDNA SynthesisTotal RNA was extracted from 19 frozen tumor and 7 control lung tissue samples using High Pure RNA tissue kit (Roche Molecular Biochemicals, GmbH, Mannheim, Germany) according to the manufacturer's recommendations. Approximately 1 μg of RNA was reverse-transcribed using oligo-p(dT)15 priming and avian myeloblastosis virus (AMV) reverse transcriptase [First Strand cDNA synthesis kit for reverse transcriptase-PCR (AMV), Roche Molecular Biochemicals] at 42°C for 1 hour. cDNAs were then stored at −20°C until further use.Real-Time Quantitative PCRHLA-G-specific PCR amplifications were performed using the following primer sets: U522(exon 3) and L922 (exon 5) detecting full-length membrane-bound HLA-G1 isoform; G5U522 (exon 3) and G5L990 (intron 4) amplifying full-length soluble, HLA-G5 isoform (Table 2.). Design of these two primer pairs and optimization for the use in LightCycler quantification system was done with the Oligo 5.0 primer analysis software (Molecular Biology Insights Inc., Cascade, CO). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and plasmids pCRII.GAPDH were kindly provided by John Gribben (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). A Hot-Start PCR was performed using 2 μl of ready-to-use mastermix (LightCycler-Faststart DNA Master SYBR Green I, Roche Molecular Biochemicals, containing thermostable recombinant Taq polymerase, reaction buffer, dATP, dCTP, dGTP, dUTP), 0.5 μmol/L of each oligonucleotide primers (desalted PCR grade; Microsynth, Balgach, Switzerland), variable free MgCl2 concentrations (Table 2), 2 μl of cDNA, and water to a final volume of 20 μl. After an initial denaturation of 7 minutes to activate the FastStart enzyme, amplification occurred as a three-step cycling procedure: denaturation at 95°C for 15 seconds, ramp rate 20°C/second; annealing (Table 2), 10 seconds, ramp rate 20°C/second; and elongation at 72°C (Table 2), ramp rate 2°C/second, for 40 cycles. The acquisition of fluorescence was done at 87°C to avoid contribution of nonspecific products to the overall signal. Finally, the temperature was raised gradually (0.2°C/second) starting from 70 to 99°C for the melting curve analysis.Table 2Oligonucleotides and PCR Conditions UsedPrimersAmplicon size, bpAnnealing °t, elongation timeFree MgCl2U522 (exon 3)5′-CAA TGT GGC TGA ACA AAG GA-3′L922 (exon 5)5′-CCA GCA ACG ATA CCC ATG-3′42060°C; 25 s4 mmol/LG5U522 (exon 3)5′-CAA TGT GGC TGA ACA AAG GAG AG-3′G5L990 (intron 4)5′-ACC GAC CCT GTT AAA GGT CTT-3′45058°C; 25 s3 mmol/LGAP1U195′-GAA GGT GAA GGT CGG AGT C-3′GAP206L195′-GAA GAT GGT GAT GGG ATT T-3′22455°C; 10 s4 mmol/L Open table in a new tab External standards for the GAPDH quantification consisted of six serial 1:10 dilutions (5 × 102 to 5 × 107 molecules per reaction) of pCRII.GAPDH plasmids. Plasmids containing the full-length HLA-G insert, pLNCX.G132Kirszenbaum M Moreau P Gluckman E Dausset J Carosella E An alternatively spliced form of HLA-G mRNA in human trophoblasts and evidence for the presence of HLA-G transcript in adult lymphocytes.Proc Natl Acad Sci USA. 1994; 91: 4209-4213Crossref PubMed Scopus (213) Google Scholar were used as six external standards for HLA-G1 and HLA-G5 quantitative PCR, containing 102 to107Rees RC Mian S Selective MHC expression in tumours modulates adaptive and innate antitumour responses.Cancer Immunol Immunother. 1999; 48: 374-381Crossref PubMed Scopus (79) Google Scholar copies per reaction (a kind gift from D. E. Geraghty, Fred Hutchinson Cancer Research Center, Seattle, WA). External standards were run concomitantly with patient samples under identical conditions. The PCR reactions were run as triplicates, and run data were analyzed with the quantification program V3.39 (Roche Molecular Biochemicals). The fluorescence signal was plotted against the cycle number for all samples and external standards. The fit-points method option was used in the course of analyzing quantification data, allowing the definition of a noise band and subsequent background fluorescence subtraction, and resulting in the display of only log-linear and plateau amplification phases. The point when the signal rose above the background level, so-called “crossing point” was then determined for each standard dilution. The standard curve was then generated for each run, plotting the crossing point against the log concentration of the standards. The single HLA-G1, HLA-G5, and GAPDH standard curves as a result of triplicate runs were then used as references to calculate the number of target molecules in each sample. Results were expressed initially as the absolute copy number/μl. Normalization of the estimated HLA-G1 or HLA-G5 amount was achieved by calculating the ratios between HLA-G1 or HLA-G5 and GAPDH copy number in 1 μl of cDNA, to compensate the variations in quantity and quality of starting mRNA. The normalized values were then multiplied by the constant 104.Each amplification product was analyzed for appropriate length by electrophoresis of on 1.6% agarose gel stained with ethidium bromide. The estimated size of the amplified fragments matched the calculated size. In addition product identity was confirmed by melting curve analysis, an application in the LightCycler analysis program. The specificity of the obtained PCR products was finally confirmed by sequencing.ImmunohistochemistryIn all 34 cases, formalin-fixed paraffin-embedded material was available for immunohistochemistry. After microwave antigen recovery an alkaline phosphatase-anti-alkaline phosphatase technique with the following primary antibodies (Abs) was performed: 4H84 (1:500 dilution), IgG1 mouse monoclonal (mAb) raised against denatured HLA-G α1-domain (kindly provided by M. McMaster, University of California, San Francisco, CA); 16G1 (1:250) IgG2a mouse mAb detecting soluble HLA-G (kindly provided by D. E. Geraghty, Fred Hutchinson Cancer Research Center, Seattle, WA); anti-MHC class I mAb TP25.99, reacting with HLA-A, -B, -C, but not HLA-G (kind gift from S. Ferrone, Roswell Park Cancer Institute, Buffalo, NY); anti-human IL-10 (E-10) mouse IgG2b mAb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-human CD56 mouse IgG1 mAb (Novocastra Laboratories Ltd., Newcastle, UK); isotype-matched controls IgG1, IgG2a, and IgG2b (DAKO, Glostrup, Denmark).Double stainings were performed for IL-10 and CD56 against the mAb 4H84 (1:300 dilution). Briefly, after deparaffinization and antigen retrieval, nonspecific binding sites were blocked by incubating slides with 20% AB serum/phosphate-buffered saline for at least 15 minutes at room temperature. Tissue sections were incubated with different primary antibodies or isotype-matched control and secondarily, always with 4H84 mAb for 60 minutes. Each antibody application was followed by two cycles of sequential incubations with rabbit anti-mouse IgG and alkaline phosphatase-anti-alkaline phosphatase-complexes (DAKO). The immunoreaction was visualized with developing solutions, containing blue-purple 5-bromo-4-chloro-3-indoxyl phosphate with nitro-blue-tetrazolium-chloride (BCIP/NBT, from DAKO), which labeled primary antibody and red neufuchsin (DAKO) marking HLA-G. Finally, sections were counterstained with hematoxylin. All of the incubations were performed at room temperature in a moist chamber.Statistical AnalysisStatistical analysis was performed using a SPSS statistical software (version 10.0; SPSS, Inc.)ResultsDifferential Expression of HLA-G mRNA IsoformsQuantitative reverse transcriptase (RT)-PCR analysis revealed that all tumor samples had detectable levels of both HLA-G1 and HLA-G5 transcripts (Figure 1). However, uninvolved lung tissue specimens transcribed preferentially full-length membrane-bound isoform HLA-G1 (Figure 1). Tumor tissue samples displayed an average of a 2.6-fold increase in HLA-G1 (t-test, P = 0.047) and a fourfold increase in HLA-G5 transcription (t-test, P = 0.016) when compared to unaffected lung samples (Figure 1). The transcriptional level for soluble HLA-G isoforms was significantly lower than for the membrane bound (t-test, P = 0.01). Variable HLA-G transcription within the samples as determined by quantitative PCR however did not correlate to the tumor histology.Tumors of High-Grade Histology Preferentially Express HLA-G ProteinImmunohistochemistry detected HLA-G in 9 of 34 tumors (26%), of which 6 (18%) were also positive for sHLA-G protein (Table 1 and Figure 2; A to C). HLA-G positivity for membrane-bound isoform (mAb 4H84) and soluble HLA-G isoform (mAb 16G1) was heterogeneous, ranging from single cells to larger clusters of positive cells (Figure 2, A to D).Figure 2Immunohistochemistry of lung tumors and adjacent lung tissue. A: Expression of membrane-bound HLA-G (mAb 4H4) on a large-cell carcinoma, T3. B: Heterogeneous membrane-bound HLA-G expression on a large-cell carcinoma, T17. C: Expression of soluble HLA-G (mAb 16G1) on a large-cell carcinoma, T3. D: Double staining for IL-10 (black chromogen) and membrane-bound HLA-G (red chromogen) in a large-cell carcinoma showing HLA-G-positive tumor cells co-expressing IL-10 (violet chromogen), T3. E: HLA-G-expressing tumor cells associated with IL-10-positive infiltrating lymphocytes, T8. F: Induction of membrane-bound HLA-G on residual lung tissue infiltrated by the tumor, T6. G: Induction of membrane-bound HLA-G on lung tissue adjacent to the tumor. H: Negativity in lung tissue distant to the tumor. I and J: HLA-G expression (I) and partial loss of MHC class I expression (J) on tumor cells on corresponding tumor sections, T17. K and L: Double staining for CD56 (black chromogen) and membrane-bound HLA G (red chromogen) showing close association of NK cells with intact HLA-G-expressing tumor cells, T18. Original magnifications: ×200 (A, E, G, H, and K), ×100 (B), ×400 (C and D), ×50 (F, I, and J), ×960 (L).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In HLA-G-positive tumors, the loss of HLA class I immunoreactivity was a consistent finding (Figure 2, H and I). In six of nine cases we detected complete loss of HLA class I molecules and focal loss in the remaining three cases (Table 1). Conversely, tumor-unaffected lung displayed no detectable HLA-G protein expression, whereas positivity for HLA class I antigens was found as expected (Figure 2, G to I). Immunoreactivity with 4H84 was mainly detected on large-cell carcinomas (Spearman's rho = 0.529, P = 0.001) and on the tumors of high-grade histology (Spearman's rho = 0.489, P = 0.004) (Figure 2, A and B). Accordingly, the expression of sHLA-G was primarily encountered in large-cell carcinomas (Spearman's rho = 0.582, P < 0.001) and in high-grade tumors (Spearman's rho = 0.495, P = 0.003) (Figure 2C). In addition, sHLA-G protein expression correlated with HLA-G5 transcriptional levels (Spearman's rho = 0.455, P = 0.05).NK Cell Infiltrate in the Absence of HLA Class I ExpressionNK cells infiltrating the tumor margin were detected by immunohistochemistry in 18 (53%) of the tumor samples. In seven of the cases NK cells were found adjacent to HLA-G-positive tumor cells (Figure 2, J and K). NK cell infiltration associated with focal or complete loss of HLA class I molecules on tumor cells (Spearman's rho = −0.338, P = 0.05) (Table 1).HLA-G Protein Up-Regulation in Tumor-Associated Lung TissueApart from the tumor cells positivity, two additional patterns of HLA-G expression could be observed. Within the tumor, residual alveolar structures displayed HLA-G immunoreactivity in the majority [20 (59%)] of the cases (Figure 2E). The second observed pattern consisted of HLA-G expression in tumor surrounding the lung parenchyma in 16 of 34 cases (Figure 2, E and F). In eight of these cases HLA-G positivity of the neighboring lung could be observed, although the tumor was HLA-G-negative (Figure 2E). Additionally, tumor-infiltrating lymphocytes were found to be positive for HLA-G expression in 10 (29%) cases. Macrophages and dendritic cells displayed occasional HLA-G immunoreactivity, and positivity was independent of the tumor histological type (data not shown).IL-10 Expression Coincides with HLA-G Protein Up-RegulationInterleukin-10-producing cells were detected in 15 (44%) cases, where HLA-G was either expressed by the tumor in 7 of 9 cases (Spearman's rho = 0.407, P = 0.017). The tumor cells and tumor-infiltrating lymphocytes were the source of IL-10 (Figure 2D). IL-10-producing cells were often localized in the vicinity of the HLA-G-positive cells (Figure 2D).DiscussionPubl

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