Bone Marrow Transplantation (2000) 25, Suppl. 2, S88–S95  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Functional deficiencies of components of the MHC class I pathway in human tumors of epithelial origin

K Delp1, F Momburg2, C Hilmes1, C Huber1 and B Seliger1

1The Johannes Gutenberg-University, IIIrd Department of Internal Medicine, Mainz; and 2The German Cancer Research Center, Heidelberg, Germany

Summary: expressed ␤ subunits X, Y and Z of the proteasome3,4 are replaced by the IFN-␥ regulated subunits LMP2, LMP7 and An association between oncogenic transformation and LMP10,4,5 thereby altering the proteolytic production of repression of different components of the MHC class I certain .6 In addition, the binding of the machinery (APM) have been activator PA28 to the proteasome also influences both the described in murine model systems. In order to discover quality and quantity of peptides.6 The yielded peptides are whether a similar correlation exists, human tumor cell translocated across the (ER) mem- lines of distinct histology with altered ras were brane via the heterodimeric complex consisting of the ATP- analyzed for the expression of APM components utiliz- dependent transporter associated with antigen processing ing RT-PCR and Western blot analyses. A hetero- , TAP1 and TAP2.7,8 In the ER, the peptides bind to geneous expression pattern of MHC class I , the MHC class I molecules and the ternary complex is TAP transporter, proteasome subunits, protea- exported through the secretory pathway to the cell some activator PA28 and the chaperones , cal- membrane. ␤ reticulin as well as was displayed by these tumor The assembly of the MHC class I heavy chain with 2- ␤ cell lines. Single or combined deficiencies in the microglobulin ( 2-m) and peptide is assisted by transient expression and/or function of TAP, LMP2, LMP10 and interactions with a number of ER-resident chaperones, such tapasin were demonstrated in 11 of 12 cell lines studied, as calnexin, , protein disulfide isomerase and the ␤ 9 whereas the expression of calnexin, calreticulin, 2- newly identified tapasin. The heterodimer microglobulin, LMP7 and PA28␣ was unaltered or only aggregates with TAP and forms a ‘large TAP complex’ weakly decreased. The impaired expression of TAP, consisting of TAP1, TAP2, MHC class I heavy and light LMP subunits and tapasin was not associated with alt- chains, calreticulin and tapasin.10,11 Tapasin is bridging ered ras, but resulted in reduced MHC class I surface TAP and MHC class I molecules thereby playing a critical expression. In particular, a significant allele- and locus- role in the MHC class I assembly. Binding of high affinity specific downregulation of the HLA-A and HLA-B peptides to MHC class I molecules leads to the dissociation haplotypes was found. IFN-␥ treatment corrected the of this ‘large TAP complex’ and the exit of the ternary TAP, LMP and tapasin deficiencies and enhanced the MHC class I molecules from the ER.11 constitutive PA28␣, LMP7, calnexin and calreticulin Studies on mutant cell lines or on knock out mice have expression which was accompanied with increased levels been extremely useful in defining the role of the different of MHC class I antigens. Thus, dysregulation rather components of the MHC class I antigen processing and than structural alterations of different APM compo- presentation pathways.12–14 Although TAP and LMP sub- nents might be one mechanism of colon carcinoma, units are not absolutely required for MHC class I antigen small cell lung carcinoma and pancreatic carcinoma cell presentation,12,15,16 their deficient expression often results lines to evade immune recognition. Bone Marrow Trans- in reduced levels of MHC class I surface molecules and plantation (2000) 25, Suppl. 2, S88–S95. impaired MHC class I restricted responses.13,15,16 Keywords: chaperones; IFN-␥; MHC class 1; peptide Loss or reduced expression of MHC class I molecules is transporter; proteasome a frequent event in human tumors of different histology and has been attributed to structural alterations and/or dysregul- ation of various components of the MHC class I antigen processing machinery (APM).17–19 A number of studies Short break-down products of cytosolic are demonstrated a coordinated TAP and LMP downregulation, presented in the context of the major but deficiencies in a single APM component have also been complex (MHC) class I molecules to cytotoxic T lympho- described.19–21 In a murine model of oncogenic transform- 1,2 cytes (CTL). The process of peptide generation requires ation, reduced MHC class I surface expression was the multicatalytic proteasome complex. The constitutively inversely associated with mutated ras oncogene expression due to suppression of TAP1 and LMP2.22 Correspondence: Dr B Seliger, Johannes Gutenberg-University, IIIrd The expression pattern of the different APM components Department of Internal Medicine, Langenbeckstr. 1, 55101 Mainz, has not been analyzed in detail in human tumors with Germany mutant ras. Therefore, we examine the constitutive and Impaired antigen processing in human tumors K Delp et al S89 (IFN)-␥ regulated expression of various chap- Immunofluorescence analysis erones, TAP, different proteasome subunits and MHC class Immunofluorescence analysis was performed using the fol- I antigens in small cell lung carcinoma (SCLC), pancreatic lowing monoclonal (mAb): MsIgG (negative carcinoma and colon carcinoma cell lines. control; Coulter-Beckman, Krefeld, Germany), the hybridoma supernatant of W6/32 recognizing HLA-A, -B, ␤ -C locus products complexed to 2-m, the locus-specific Materials and methods mAb HLA-A and HLA-B kindly provided by Dr S Ferrone (Valhalla, USA), the allele-specific mAb recognizing HLA- A1, HLA-B7 and HLA-A2.1 (BB7.2), mAb 148.3 reco- Cell lines, cell culture and IFN-␥ treatment gnizing TAP1, and BBM1, which was kindly provided by E Weiss (Institute of Human Genetics, Mu¨nchen, Germany), With the exception of Panc1 and Stom1 the human tumor ␤ recognizing free and complexed 2-m. The second cell lines of distinct histology employed in this study were was FITC-labeled goat anti-mouse antibody (Coulter- obtained from the American Tissue Type Culture Collec- Beckman). tion (Rockville, MD, USA). The HLA phenotype of the Prior to staining with the mAb 148.3 directed against different cell lines, kindly provided by E Hilmes (Johannes TAP1, cells were permeabilized with 70% ethanol for Gutenberg-University, Blood Bank) was determined by 30 min on ice. For cytofluometry, 5 ϫ 105 to 1 ϫ 106 cells genomic polymerase chain reaction (PCR) using the Dynal were incubated for 30 min at 4°C with an excess of the SSP primer system (Dynal, Hamburg, Germany). All tumor respective antibody, washed twice with PBS, incubated cell lines were grown in Dulbecco’s medium (Biochrom, with FITC-GAM for an additional 30 min at 4°C before Berlin, Germany) supplemented with 10% fetal calf serum being analyzed on a flow cytometer (Coulter EPICS XL, (FCS) (Greiner, Frickenhausen, Germany), 2 mm gluta- MCL, Coulter-Beckman). mine, penicillin and streptomycin. The human lympho- blastoid cell line T1 and its mutant derivative T223 encompassing deletions of the TAP1/2 and LMP2/7 genes, Peptide synthesis and peptide binding analysis were maintained in CRPMI (Biochrom) completed with The following peptides synthesized on solid phase using F-

10% FCS, glutamine and respective antibiotics and served moc for transient NH2-terminal protection and charac- as controls. terized by amino acid analysis were employed in peptide Cells were treated with human recombinant 50 ␮g/ml binding assays: the HLA-A2 binding HIV peptide C7 (HIV IFN-␥ (specific activity of 3 ϫ 106 U/mg protein) kindly pol 510–518; ILKEPVHGV) and the H-2 binding CMV provided by Thomae (Biberach, Germany) for the indicated peptide A7 (YPHFMPTNL). Briefly, 5 ϫ 105 cells were length of time. incubated for 15 to 18 h in serum-free medium in the pres- ence or absence of 100 ␮m peptide with or without ␮ ␤ 2.5 g/ml human 2-m (Sigma, St Louis, MO, USA). Primers, RT-PCR analysis and sequencing Peptide-labeled cells were stained by indirect immuno- fluorescence analysis as described.20 The oligonucleotides used for PCR analysis, sequencing and hybridizations were generated by employing the Primer Peptide translocation assay Designer program (Version 2.01; Scientific & Educational Software), synthesized by the triester method and pur- For peptide translocation, 2.5 ϫ 106 cells/incubation were chased from MWG Biotech (Ebersberg, Germany). The pri- harvested and washed once with incubation buffer (130 mm mers and the PCR conditions used are listed in Table 1. KCl, 10 mm NaCl, 1 mm CaCl2,2mm EGTA, 2 mm 7 25 Total cellular RNA from 5 ϫ 10 cells was isolated by the MgCl2,5mm Hepes, pH 7.3). Cells were permeabilized guanidinium isothiocyanate method and subjected to one- with 2 IU streptolysin O/ml (Welcome Reagent, Becken- step RT-PCR analysis (Titan Kit; Roche Diagnostics, ham, UK) in 50 ␮l incubation buffer for 10 min at 37°C Mannheim, Germany) as recently described.24 In most followed by the addition of 10 ␮l ATP (10 mm; Roche cases, ␤-actin and APM specific PCR was performed in the Diagnostics), 10 ␮l radioiodinated peptide and additional same reaction using 10 cycles of 20 s at 94°C at the incubation buffer to a final volume of 100 ␮l. Peptide trans- annealing temperature, 1 min at 68°C followed by 25 cycles location was inhibited by lysing the permeabilized cells of 1 min at 94°C, 1 min at the annealing temperature, 1 min with 1 ml NP-40 lysis mix. Glycosylated peptides were at 68°C with a cycle elongation of 5 s for each cycle. recovered with ConA-Sepharose (Pharmacia, Uppsala, The amplification products were size fractionated on a Sweden) as described.25 1% agarose gel and the specificity of analysis was con- firmed by determination of the predicted size as well as by Southern blotting of the gels followed by processing of fil- Results ters with digoxygenin (DIG)-labeled specific oligonucleo- Reduced expression of MHC class I antigen processing tides using the DIG chemiluminescence detection kit genes in human tumors of epithelial origin is not (Roche Diagnostics). expression was quantitated by associated with ras mutations densitometric analysis of the autoradiograms and nor- malized to the ␤-actin signal. Mutations of the ras oncogene In order to evaluate whether a relationship between ras were evaluated by directly sequencing the PCR products.24 mutations/overexpression and suppression of the MHC

Bone Marrow Transplantation Impaired antigen processing in human tumors K Delp et al S90 Table 1 Sequences and conditions of primers used for PCR analyses

Molecule Primers from 5Ј to 3Ј Annealing temp. Number of cycles Oligonucleotides used for hybridization in °C

TAP1 f GAGACATCTTGGAACTGGAC 59 40 GTCGGCCATGCCTACAGTTCGAAGCTTTGC b CTCTGAGTGAGAATCTGAGC TAP2 f TTCATCCAGCAGCACCTGTC 57 35 GCTGTATTGGCGGAGAGACCT b CTCTTGCGAAGCCTGGTGAA LMP2 f CAGCTGTAATAGTGACCAGG 60 35 AGAGGACTTGTCTGCACATCTCATGGTAGC b CATCTACTGTGCACTCTCTG LMP7 f GCCACATGAGTGTCTTACTG 64 35 ACCTCACCTCTACAGCATCTATCCTCATGG b CAGACACAGACATGACAACC LMP10 f GATACGCGAGCCACTAACGA 59 35 CTGATCGTGGGCGGCGTAGACCTGACTGGA b GTAGCGGCCAGACCTCTTCA PA28␣ f GCATCACCACGCTCAGAGAA 54 25 GTGGCCCAGTGAACTGCAATGAAAAGATCG b GGAGCCAGCTCTCAATGAAG calreticulin f CTCTCCGTCCATCTCTTCAT 54 35 CCTATGAGGTGAAGATTGACAACAGCCAGG b AAGTTCTACGGTGACGAGGA calnexin f TTCACATAGGCACCACCACA 53 30 TTGACGATGTCATTGAAGAGGTAGAAGACT b GGAAGTGGTTGCTGTGTATG tapasin f CATGCTGCTCAGTGGTGACTGG 61 40 GCCTCAGCAGGAGCCTGTTCTCATCACCAT b CGCGGTGATCGAGTGTTGGTTC ␤ 2-microglobulin f CTCGCGCTACTCTCTCTT 57 30 AAGCTATCCAGCGTACTCCAAAGATTCAGG b AAGACCAGTCCTTGCTGA

f = forward; b = backward.

class I antigen processing pathway exists, the ras phenotype (Table 3, Figure 1), whereas others are only marginally sup- of 12 human tumor cell lines of distinct origin was analyzed pressed or are even unaltered. Calnexin and calreticulin (Table 2). Seven out of 12 tumor cell lines possessed a were expressed in all tumor cell lines and the level of mutation in codon 12;13 of the Ki-ras gene resulting in an expression was similar to that of T1 cells, whereas tapasin amino acid (aa) change from glycin to asparagin, arginin, expression was only strongly decreased in the SCLC cell valin or serin, respectively. No structural alterations were line Colo 699. Thus, alterations in the constitutive chap- found in the Ki-ras codon 61 and in the mutation hot spots erone expression were limited to tapasin and only to a min- ␤ of the N-ras gene of these tumor cells (data not shown). ority of tumor lines. In addition, LMP7 and 2-m transcrip- The mRNA expression of APM components in the tumor tion was not altered in any tumor cell line tested. With cell lines with variable ras phenotype as well as T1 cells the exception of Colo 699 cells, PA28␣ transcription was and pbmnc encompassing normal expression or charac- comparable in all tumor cell lines to that of respective terized deficiencies (T2 cells) was analyzed by RT-PCR control cells. using ␤-actin primers as the internal standard. Deficient or The most prominent differences were obtained by analy- reduced expression of certain genes was found in a high ses of the LMP2, LMP10, TAP1 and TAP2 transcription. percentage of colon, SCLC and pancreatic tumor cell lines Five out of 12 tumor cell lines displayed impaired TAP1

Table 2 Characteristics of tumor cell lines

Cell line Origin HLA phenotype Ki-ras codon Ki-ras AA code MHC class I 12;13 wt: Gly;Gly expression (MFI) A-locus B-locus C-locus wt: GGT;GGC; without/with IFN␥

Stom stomach carcinoma 24 *5201 *12 GGT;GGC; Gly;Gly 20/30 Panc pancreatic carcinoma 1,11 8,35 w4,w7 GR*T:GGC: Gly/Asp;Gly 65/161 818.4 pancreatic carcinoma 2,11 40,44 w5,w10 CGT;GGC; Arg;Gly 19/168 ASPC.1 pancreatic carcinoma 1,26 15 w10 GAT;GGC Asp;Gly 13/35 CX 1 colon carcinoma 1,24 35,44 w4 GGT;GGC; Gly;Gly 13/54 Colo 206F colon carcinoma 1,2 7,8 w7 GGT;GGC; Gly;Gly 36/174 Colo 320 colon carcinoma 24 *1402 w8 GGT;GGC; Gly;Gly 7/66 Colo 678 colon carcinoma 2 7 w7 GR*T;GGC; Gly/Asp;Gly 22/102 DLD 1 colon carcinoma 2,24 8,35 w4,w7 GGT;GAC; Gly;Asp; 1/1 SW 480 colon carcinoma 2,24 7,12 w7 GTT;GGC; Val;Gly 28/81 A 549 lung carcinoma 25,30 18,44 w*12,w*16 AGT;GGC; Ser;Gly; 28/118 Colo 699 lung carcinoma 2 7,57 w6,w7 GGT;GGC; Gly;Gly 33/167

The HLA and ras phenotype of tumor cells was assessed as described in Materials and methods. MHC class I surface expression of untreated and IFN- ␥ treated (48 h, 50 ng/ml IFN-␥) cells was determined by flow cytometry using the mAb W6/32. The results are expressed as mean specific fluorescence intensity (MFI). The MFI of T1 cells, serving as positive control, was 85/197. Bold letters represent the mutations in ras; asterisks: not clearly defined.

Bone Marrow Transplantation Impaired antigen processing in human tumors K Delp et al S91 Table 3 Heterogeneous expression pattern of antigen processing genes in tumor cells of distinct origin

␥ ␣ ␤ Cell lines IFN- TAP 1 TAP 2 LMP 2 LMP 7 LMP 10 PA 28 CalreticulinCalnexin Tapasin 2-m pbl Ϫ (ϩ)(ϩ) ϩϩ ϩ ϩϩ ϩϩ ϩ ϩϩ ϩ ϩϩ T1 Ϫϩϩϩϩϩϩϩϩϩϩϩ ϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ T2 ϪϪϪϪϪϩϩϩϩϩϩϩϩ ϩϪϪϪϪϩϩϩϩϩϩϩϩϩϩϩϩ Stom Ϫ (ϩ)(ϩ)(ϩ) ϩ ϩϩϩϩϩϩ ϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩϩ Panc Ϫϩ(ϩ) ϩϩϩϩϩϩ(ϩ) ϩϩ ϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ 818.4 Ϫϩ(ϩ) ϩ ϩ ϩϩϩϩϩϩ ϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ ASPC.1 Ϫϩ(ϩ) ϩ ϩ ϩϩϩϩϩϩϩ ϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩ ϩϩϩ CX 1 Ϫ (ϩ)(ϩ)(ϩ) ϩ ϩϩϩϩϩϩ ϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ Colo 206F Ϫϩϩϩϩϩϩϩϩϩϩ ϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ Colo 320 Ϫ (ϩ) ϩ (ϩ) ϩ (ϩ) ϩϩϩϩϩ ϩ ϩϩϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩ ϩϩϩ Colo 678 Ϫϩ(ϩ) ϩ ϩ ϩϩϩϩϩϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ DLD 1 Ϫ (ϩ) ϩ (ϩ) ϩ (ϩ) ϩϩϩϩϩ ϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩ ϩϩ ϩϩ ϩϩϩ SW 480 Ϫϩ(ϩ) ϩϩϩϩϩϩϩϩϩ ϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ A 549 Ϫ (ϩ)(ϩ) ϩ ϩ ϩϩϩϩϩϩ ϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ Colo 699 Ϫϩ(ϩ)(ϩ) ϩϩ(ϩ) ϩϩ(ϩ) ϩ ϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ pbl = peripheral blood mononuclear cells. Cell lines were left either untreated or treated with IFN-␥ (50 ng/ml, 24 h) before RNA extraction followed by RT-PCR was performed as described in Materials and methods.

Figure 1 Expression of APM genes in human tumor cell lines. RNA was prepared from cells grown in the presence and absence of IFN-␥ (50 ng/ml; 24 h) prior to RT-PCR analysis. The amplification products were size fractionated on agarose gels and specificity of products was examined by either ␤ ethidium bromide staining or Southern blot hybridization of the gel. RT-PCR with 2-actin served as controls. and LMP2 expression, TAP2 was suppressed in 9/12 tumor (Table 3; Figure 1). However, a relationship between cell lines and LMP10 in 2/12 tumor cell lines. In Stom and downregulation of TAP/LMP and ras mutations did not CX1 cells, the expression of TAP1, TAP2 and LMP2 was exist. coordinately downregulated. A simultaneous suppression of TAP1 and LMP2 transcription was found in CX1 and A549

Bone Marrow Transplantation Impaired antigen processing in human tumors K Delp et al S92 Decreased expression of peptide transporters is MHC class I surface molecules in APM-deficient tumor associated with impaired function but not with the ras cell lines could not be stabilized at physiological phenotype temperature or by MHC class I binding peptides In order to analyze whether reduced TAP transcription MHC class I surface expression of APM-defective cells can affects TAP activity, peptide translocation assays were be stabilized both by culture at low temperature or by the performed in the presence and absence of ATP utilizing addition of specific HLA-binding peptides.26 Peptide bind- two different input peptides. Representative results with ing assays were performed with the specific HLA-A2 bind- the peptides #600 (TNKTRIDGQY) and #63 ing HIV peptide and the nonsense H-2 binding CMV pep- (RYWANATRSI) demonstrated an ATP-dependent peptide tide as negative control. Incubation with the HLA-A2- transport (data not shown) which is highly variable in the specific binding peptide also did not influence the level of different tumor cell lines analyzed (Figure 2). The transport MHC class I surface expression in HLA-A2ϩ tumor cells of peptide #600 was in most cases more effective than that (Figure 4, upper panel), whereas MHC class I expression of peptide #63 ranging from 0.4% (negative) to more than of T2 cells was three-fold increased. As expected, the CMV 14% in the pancreatic carcinoma cell line ASCP1 and the control peptide did not affect MHC class I surface SCLC line A549. TAP activity was totally abrogated with expression. These results were further confirmed by tem- a transport rate for both peptides of 0.4 to 0.6% in CX1 perature assays. The cell lines were in parallel maintained cells, which was comparable to that of TAP-deficient T2 for 36 h at 26°Cor37°C before MHC class I surface cells (data not shown). expression was determined. T1 cells and their APM- deficient mutant T2 served as controls. MHC class I expression was not enhanced by incubation tumor cells and Locus-specific downregulation of MHC class I surface T1 cells at low temperature, whereas enhancement of MHC expression in human epithelial tumor cell lines class I surface expression was observed on mutant T2 cells (Figure 4, lower panel), suggesting that deficient expression MHC class I surface expression of the various tumor cell and function of APM genes is only partially responsible for lines was determined by flow cytometry using a mAb lack or reduced MHC class I surface expression of the (W6/32) recognizing human HLA class I antigens, the tumor cell lines analyzed. locus-specific mAb HLA-A and HLA-B as well as the allele-specific mAb HLA-B7 and mA2.1 recognizing HLA- Treatment with IFN-␥ caused reversion of most antigen A2. Staining with mAb W6/32 revealed a significantly processing deficiencies reduced MHC class I surface expression in pancreatic, colon and small cell lung carcinoma cell lines when com- IFN-␥ is known to induce the expression of most compo- pared to the lymphoblastoid T1 cells (Table 2). DLD1 cells nents of the antigen processing pathway such as TAP, even totally lack MHC class I surface expression. Rep- LMP, PA28 and MHC class I molecules.19,27 Therefore, the resentative results were shown for Colo 699, CX1 and Colo effect of IFN-␥ on the described APM deficiencies in colon, 206F cells (Figure 3), further demonstrating a strong HLA- pancreatic, stomach and lung carcinoma cells was deter- A and HLA-B locus and/or allele-specific downregulation mined. IFN-␥ treatment of these tumor cell lines results in when compared to total MHC class I surface expression. an approximately two-fold induction of the mRNA

Figure 2 TAP-dependent peptide translocation of model peptides. The radiolabelled peptides #63 (RYWANATRSI white bars) and #600 (TNKTRIDGQY black bars) were incubated in the presence and absence (data not shown) of ATP with streptolysin O permeabilized tumor cells. The translocated peptides were isolated and quantitated as described in Materials and methods. The results represent the mean of two experiments.

Bone Marrow Transplantation Impaired antigen processing in human tumors K Delp et al S93

Figure 3 Total locus- and allele-specific down-regulation of MHC class I surface expression. Different human tumor cells incubated in the absence and presence of IFN-␥ (50 ng/ml, 48 h) were analyzed for MHC class I surface expression using a panel of total, locus- and allele-specific mAb by flow cytometry. The results represent the mean specific fluorescence intensity. expression of all APM components, including HLA class I of tumor cell lines analyzed displayed a simultaneous antigens, TAP, LMP, PA28␣, the chaperones tapasin, cal- downregulation of multiple APM components spanning nexin and calreticulin (Figure 1 and Table 3), which was different loci, whereas PA28 expression was not down- also associated with increased TAP activity and enhanced regulated when compared to control cells. total, locus- and allele-specific MHC class I surface Our experiments evaluated a panel of human tumor cell expression (Figure 3). Thus, the deficiencies in the antigen lines of different histology with distinct ras phenotypes for processing pathway could be reverted by cytokine treatment the expression pattern of TAP, LMP subunits, PA28, cal- and therefore seemed to be due to dysregulation rather than nexin, calreticulin and tapasin. Deficiencies in the structural alterations. expression of single or multiple APM component(s) were detected in 11 out of 12 tumor cell lines tested. The defects observed in the tumor cell lines studied did not affect all Discussion TAP and LMP genes clustered in the MHC class II region. A simultaneous suppression was not even obtained in all There exists strong evidence that one major immune escape cases for TAP1 and LMP2, which are regulated by a shared mechanism of tumors of different histology, such as SCLC, bidirectional promoter.28 However, heterogeneous TAP renal cell carcinoma, cervical carcinoma, colon cancer and transcription was directly associated with its function melanoma is the impaired expression and function of differ- (Figure 2), but the observed differences in the transport rate ent APM components, particularly TAP1, TAP2, LMP2 of peptide #600 and #63 cannot be explained yet, although and LMP7, all located within the MHC class II region.19,20 Neefjes and coauthors demonstrated variable transport Recently, Johnssen and coauthors21 extended these analyses efficiencies depending on the peptide employed.29 to the proteasome subunits LMP10, PA28␣ and PA28␤ An important role in effective antigen processing was known to be involved in the antigen processing, but enco- implicated for chaperones. For example, tapasin-defective ded outside of the MHC class II locus. In this study, 30% cells lack MHC class I surface expression, which could be

Bone Marrow Transplantation Impaired antigen processing in human tumors K Delp et al S94

Figure 4 Stabilization of MHC class I surface expression of tumor cells by different culture conditions. Upper panel: peptide binding assays. Cells

were incubated with MHC class I binding peptides (C7: HLA-A2 binding peptide; A7: nonsense peptide) in the presence and absence of ␤2-m overnight as described in Materials and methods. The control was treated with DMSO alone. MHC class I surface expression was determined by flow cytometry. Results are expressed as % induction of MHC class I molecules when compared to untreated control cells. Lower panel: Temperature shift experiments. The cells were incubated at 26°Cor37°C for 16 h prior to flow cytometry using MHC class I specific antibody. The results are expressed as % induction of mean specific fluorescence intensity of MHC class I surface expression at 37°C.

reconstituted by tapasin gene transfer.14 The analysis of may provide the cell with essential functions independent chaperone transcription demonstrated that calnexin and cal- of the effective antigen processing, although IFN-␥- reticulin expression was unaltered, but constitutive tapasin mediated induction of calnexin and calreticulin not only expression was reduced in the SCLC cell line Colo 699, argues for their general importance for cellular functions, also expressing low levels of MHC class I surface antigens. but also for immune responses. Thus, impaired tapasin expression occurs only in a minority In summary, the immune escape mechanisms of human of tumors but may play an important role for immune tumor cells may involve single or combined deficiencies of escape (Figure 1, Table 3). However, to further evaluate its different APM components, but the hierarchy of these importance, more tumors have to be monitored. events has still to be elucidated. Further knowledge of the In previous studies, an in vitro model of ras transformed underlying mechanisms of downregulation of APM may be murine fibroblasts demonstrated that ras expression was essential for the proper design of individually tailored T inversely associated with impaired MHC class I surface cell-mediated immunotherapeutical strategies. expression due to TAP1 and LMP2 downregulation.22 Until now, limited information has been available whether altered ras expression in human tumor cells affects MHC class I as Acknowledgements well as APM , thereby resulting in reduced sensitivity to CTL-mediated lysis. Our results argue against The authors would like to thank Thomas Spies and Peter a direct correlation between ras mutations and APM Cresswell for TAP and LMP plasmids, Elisabeth Weiss, Soldano deficiencies, since single or combined APM defects were Ferrone and Thomas Wo¨lfel for generous gifts of antibodies as observed in colon, pancreatic and SCLC tumor cells both well as Inge Schmidt for help in preparing the manuscript. This in the presence and absence of ras mutations (Table 2; work was supported by a grant from the SFB 432 A5 (BS) and Figure 1). However, these data have to be extended to a SFB 311 (BS). This publication represents a part of the MD thesis larger number of tumor cell lines and to in vivo tumor of K Delp. lesions. In addition, the relatively frequent loss of MHC class I molecules and TAP1 in lung cancer has to be re- evaluated concerning the ras phenotype of the APM- References defective tumor specimen.19,30 The observed impaired APM expression could mainly be 1 Zinkernagel RM, Doherty PC. MHC restricted cytotoxic T ameliorated by IFN-␥ treatment suggesting that the cells: studies on the biological role of polymorphic major deficient expression is due to dysregulation. The constitut- transplantation antigens determining T cell restriction speci- ␣ ficity, function and responsiveness. Adv Immunol 1979; 27: ive expression pattern of PA28 calnexin and calreticulin 51–77. is relatively conserved in tumor cells in comparison to 2Ro¨tzschke O, Falk K, Deres K et al. Isolation and analysis of TAP, LMP and tapasin. The major role of the latter genes naturally processed viral peptides as recognized by cytotoxic seemed to be the effective generation and transport of anti- T cells. Nature 1990; 348: 252–254. genic peptides, whereas calnexin, calreticulin and PA28␣ 3 Belich MP, Glynne RJ, Senger G et al. Proteasome compo-

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