Amplification of RNAi—Targeting HLA mRNAs

ARTICLEdoi:10.1016/j.ymthe.2004.12.023

Amplification of RNAi—Targeting HLA mRNAs

Sergio Gonzalez,1 Daniela Castanotto,2 and Haitang Li2 Simon Olivares1

Michael C. Jensen1,3,4 Stephen J. Forman4

John J. Rossi2,* Laurence J.N. Cooper1,3,4

1Division of Molecular Medicine, 2Division of Molecular Biology, 3Division of Pediatric Hematology–Oncology, and 4Division of Hematology and

Hematopoietic Cell Transplantation, Beckman Research Institute and City of Hope National Medical Center, Duarte, CA 91010, USA

*To whom correspondence and reprint requests should be addressed at the Division of Molecular Biology,

Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte,

CA 91010-3000, USA. Fax: (626) 301-8271. E-mail: [email protected].

Available online 12 February 2005

MOLECULA

Copyright C

1525-0016/$

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier tosuccessful analytic and therapeutic application. We demonstrate that increasing expression ofintroduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that thisapproach can be used to achieve maximal target down-regulation, when the choice of optimalsiRNA-binding sites is restricted or when multiple genes are simultaneously targeted and theamount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies ofshRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector.Using this system, we achieved simultaneous down-regulation of expression of classical humanleukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might beapplied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparateallografts.

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Key Words: RNA interference, siRNA, posttranslational gene silencing, short hairpin RNA,Pol III promoter, DNA plasmid, T cells, HLA, nonviral gene therapy, gene therapy

INTRODUCTION

The classical human leukocyte antigen (HLA) class Imolecules function both as alloantigens to triggerimmune recognition and graft rejection of allogeneiccells in unmatched transplant recipients and as a plat-form to present self or foreign peptides that can berecognized by CD8+ T cells bearing a clonotypic T cellreceptor [1]. It has been previously demonstrated thatintroducing viral immune evasion genes can modulateimmune recognition by blocking expression of classicalHLA class I molecules [2–5]. However, since smallinterfering RNAs (siRNAs) are a small nucleic acid reagentthat, in contrast to virally derived proteins, are unlikelyto elicit an immune response, we developed a strategy toexpress intracellular siRNAs, homologous to a sequenceconserved in most classical polymorphic HLA-A, -B, and-C loci, as hairpin transcripts from mammalian RNApolymerase III (Pol III) promoters [6,7] to achievesuppression of major histocompatibility complex(MHC) class I cell-surface expression.

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e American Society of Gene Therapy

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The data in this report demonstrate a method toamplify short hairpin (sh) RNA expression and the siRNAeffect. This was achieved by increasing the number of PolIII promoters and double-stranded (ds) RNA cassettes in aDNA expression plasmid. An application of this approachis demonstrated by down-regulating HLA gene expressionin human T cells.

RESULTS AND DISCUSSION

A design of HLA ABC-specific siRNA is constrained bychoosing a target site homologous to the majority ofclassical class I alleles, as multiple endogenous genes needto be simultaneously targeted to achieve down-regulationof HLA molecules. Since the restricted targetable sitescould be associated with adverse siRNA position effects,we developed a system to titrate/augment expression ofshRNA using a plasmid vector containing a differentnumber of U6 promoters and shRNA cassettes (Figs. 1Aand 1B). Fig. 1C depicts an HLA class I molecule and the

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FIG. 1. (A) Diagram of the HLA ABC-specific or HLA A-specific U6shRNA cassette (not to scale). The 9-nucleotide hairpin loops and 6-nucleotide terminator

sequences are shown in lowercase. (B) Schematic of DNA expression plasmids EGFP/Neo-diipMG and HyTK-pMG, modified to express multiple copies of the

U6shRNA cassettes. The EGFP gene is under control of the human elongation factor (EF) 1a hybrid promoter and NeoR and HyTK genes are under control of the

CMV IE promoter. The bovine growth hormone (bGhpA) and late SV40 poly(A) sites (SV40pA) are shown. The synthetic poly(A) and pause site (SpAn) and

Escherichia coli origin of replication are shown. (C) Schematic of HLA A3 molecule and relative binding sites of siRNA antisense strands and PCR primers (not to

scale). Signal peptide (SP); a1, 2, and 3 regions; and cytoplasmic region are shown as determined from SWISSPROT:1A03_HUMAN.

ARTICLE doi:10.1016/j.ymthe.2004.12.023

relative positions of the siRNA binding sites used in thisstudy.

To titrate/augment RNA interference (RNAi) effects,we transiently down-regulated MHC class I expression onJurkat cells, a T cell line expressing HLA A*0301/0301B*0702/3503 Cw*401/0702, transfecting with a panel ofDNA vectors containing between 0 and 6 copies of theU6shRNA cassette. A flow cytometry kinetic study dem-

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onstrated that the down-regulation of HLA ABC antigenspeaked between 3 and 4 days after transfection (Fig. 2A),reflecting the time required to achieve sufficient shRNAexpression and RNAi to prevent replacement of HLA A,HLA B, and HLA C molecules on the cell surface.Strikingly, increasing the copy number of the U6shRNAcassettes from 1 to 6 resulted in a steady increase in RNAi,with a maximal 17-fold improvement in the siRNA effect.

MOLECULAR THERAPY Vol. 11, No. 5, May 2005

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Down-regulation of HLA ABC expression was specific ascells transfected with a DNA plasmid expressing ascrambled version of the HLA ABC-specific shRNAshowed negligible loss of HLA class I cell-surface expres-sion (Fig. 2A).

We were also able to achieve durable down-regulationof HLA ABC levels as a result of augmented shRNAexpression. While expression of two copies of theU6shRNA cassettes resulted in 5.3% of the G418-resistantT cells with down-regulated protein expression of bothHLA ABC and h2-microglobulin (h2-m), this percentageincreased approximately 11-fold when 6 copies of theU6shRNA cassettes were expressed (Fig. 2B). We used ascrambled version containing 6 copies of the shRNAcassettes as a control for nonspecific effects in this (Fig.2B) and all subsequent experiments and it showed nodown-regulation of HLA ABC.

Southern blotting analyses confirmed that the G418-resistant Jurkat cells had integrated the correct number ofU6shRNA cassettes (Fig. 2C). The siRNA-mediated down-regulation of HLA ABC has been maintained for anextended period of time, as transfected Jurkat cellscontinue to demonstrate down-regulation of HLA ABCprotein expression after 6 months of passage in tissueculture (data not shown). We detected no IFN-h produc-tion, a nonspecific effect induced by expression ofshRNA, in cells expressing multiple copies of theU6shRNA cassettes (data not shown).

The degree of HLA ABC protein down-regulationcorrelated with the level of expression of stem–loopdsRNA as confirmed by Northern analyses of the shRNAconstructs (Fig. 2D). We subsequently increased thenumber of U6shRNA cassettes from 6 to 10 copies andobserved a proportional increase in shRNA down-regu-lation activity (up to 21-fold) in transiently transfectedcell lines (not shown). However, the ability to down-regulate HLA ABC protein expression in stable trans-fectants peaked with the introduction of 6 copies of theshRNA cassettes, while 7 to 10 copies showed a decreasein HLA down-regulation, which was consistent with arelative decline in their intracellular RNA expression(data not shown). The reason(s) for the loss in expressionand efficacy in stable cell lines with greater than 6 copiesof the cassette is not clear, but could include localchromatin alterations resulting in relative loss of Pol IIIexpression and/or a selective disadvantage of stableoverexpression of anti-HLA ABC shRNA. Thus, theremight be a limit to the number of U6shRNA cassettesthat can be successfully used once the vector is integratedin a specific cell line.

To exclude the possibility that, despite the similarity ofthe HLA nucleic acid sequences, there may be differentialinhibitory effects for individual MHC class I alleles(indicative of position-dependent siRNA effects), leadingto selective down-regulation of individual alleles, whichcould not be detected using a pan-HLA class I-specific



monoclonal antibody (mAb), we determined the cell-surface expression of HLA A3 and HLA B7 using allele-specific mAbs. Increasing the number of HLA ABC-specific U6shRNA cassettes resulted in a much greaterdegree of down-regulation of cell-surface HLA A3 proteinexpression relative to HLA B7 protein expression (Fig. 3A).This was surprising since Jurkat cells are homozygous forHLA A3 and appear to express more HLA A mRNA thanHLA B [8]. RT-PCR analyses of RNA extracted fromnontransfected Jurkat cells or from cells transfected withthe parental vector or the scramble U6shRNA, which wereused as controls, showed that in all cases the HLA Aexpression was at least 3-fold higher than HLA Bexpression (Fig. 3B). Given the variability observed intarget propensity to RNAi effects, it is likely that theindividual susceptibilities of HLA A and B transcripts toshRNA down-regulation differ due to the differentsequence contexts flanking the RNAi target site, whichare currently not predictable from the nucleotidesequence. Despite this possibility of adverse nucleotideposition effects, increasing the number of U6 promoterand HLA ABC-specific stem–loop cassettes from 1 to 6copies still improved the down-regulation of HLA B7 by13-fold, indicating that increasing shRNA expression canbe used as a strategy to help overcome the consequencesof target position dependency of RNAi.

To test if transfected cells that have down-regulatedclassical HLA class I molecules due to a siRNA effect arecapable of avoiding immune recognition by HLA-restricted antigen-specific ahTCR+ CD8+ cytotoxic Tlymphocytes (CTL), we isolated a Jurkat clone (1A9) withhigh expression levels of the shRNA and a consequentlow level of HLA A3 expression (Figs. 4A and 4B). Flowcytometry demonstrated that this clone had 97%reduced HLA A3 cell-surface protein expression, com-pared with cells expressing no shRNA. To test forprotection against a CTL-response, we loaded the Jurkatclone exogenously with a saturating concentration of theHLA A3-restricted peptide RLRPGGKKK (derived from thep17 subregion of HIV gag), which is recognized by theHLA A3+ cytolytic T cell clone 28A2-15 [9]. We cocul-tured the peptide-loaded 1A9 cells with 28A2-15, andafter 4 h we analyzed the expression of HLA A3 onsurviving Jurkat cells by flow cytometry. After thecoculture of T cells with peptide-loaded Jurkat clone,the percentage of EGFP+ Jurkat cells that were devoid ofHLA A3 cell-surface expression increased sevenfold from8.5 to 56% (Fig. 4C), as this assay selects for cells thathave essentially lost expression of HLA A3, given thatCTL can be activated for cytolysis by as few as 1 to 100cell-surface MHC molecules [10].

To demonstrate the activity of shRNA in primary Tcells and avoid autodeletion of T cells that had lostexpression of classical HLA class I molecules by autolo-gous NK-T cells present in PBMC, we constructed a newshRNA with a 21-nucleotide sequence completely

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homologous to most HLA A alleles, but which containedbase pair mismatches with HLA B and C alleles. Togenerate HLA A2� T cells that could be eliminated invivo by ganciclovir-mediated ablation, we transfectedheterozygous and homozygous HLA A2+ primary T cellswith the HyTK-pMG plasmid, modified to express 6copies of the HLA A-specific shRNA (Fig. 1B). Hygrom-ycin-resistant T cells could be demonstrated to havedown-regulated HLA A2 expression, relative to drug-

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resistant parental T cell controls that do not express theshRNA (Fig. 5). As expected, there was only a smalldecrease in the binding of the mAb specific for HLA ABCto the T cells that had down-regulated HLA A2 expres-sion, reflecting the fact that this mAb clone recognizedan epitope also present on HLA B and C molecules (Fig.5, inset). To our knowledge, this is the first demonstra-tion of siRNA effects in primary T cells electroporatedwith a DNA plasmid, a vector system that is currently


Copyright C The American Society of Gene Therap
y

FIG. 2. (A) Kinetic analysis of expression of multiple copies of the U6shRNA cas

expression as determined by flow cytometry analyses. Transient Jurkat transfectan

percentage loss of binding of PE-conjugated anti-HLA ABC. Analyses of HLA ex

differences in transfection efficiency. The percentage down-regulation of HLA ABC

of the scrambled U6shRNA control cassette at each time point was less than 4%

the first-order polynomial for y = 8.41x � 7.06 (R2 = 0.94). This experiment was

deviation in the EGFP down-regulation for each time point. The numbers 2, 4, a

expressed from each construct. (B) Expression of multiple copies of the U6shRN

class I protein expression. G418-resistant Jurkat cells transfected with EGFP/Neo-d

scramble control were analyzed by multiparameter flow cytometry for binding o

( y axis), noncovalently expressed with soluble h2-microglobulin on the cell su

percentage of cells in the lower left quadrant (HLA ABClowh2-mlow) is shown fo

bearing U6shRNA cassettes. G418-resistant genetically modified Jurkat cells we

U6shRNA cassette copy number is indicated at the top. (D) Northern blot analys

Jurkat cells transfected with up to 6 copies of the U6 promoter and HLA ABC-spe

strand of the shRNA, are shown. An oligonucleotide complementary to the endog

The U6shRNA cassette copy numbers are indicated at the top.

FIG. 3. (A) Augmented siRNA expression from multiple U6shRNA cassettes

can help overcome adverse siRNA position effects. Expression of HLA A3 and

B7 on G418-resistant Jurkat cells transfected with EGFP/Neo-diipMG plasmid

containing 0 to 6 copies of the HLA ABC-specific shRNA cassettes or their

corresponding shRNA scramble controls is shown. The number of the

expressed U6shRNA cassettes is indicated at the x-axis. Transfectants were

analyzed by multiparameter flow cytometry for binding of biotin-conjugated

anti-HLA A3 and anti-HLA B7 on EGFP+ cells. The percentage down-regulation

of HLA A3 and B7 of Jurkat cells transfected with EGFP/Neo-diipMG plasmid

expressing 6 copies of the scrambled U6shRNA control cassettes (HLA A3 scr

and HLA B7 scr) at each time point was less than 4% and it is shown at day 6.

This experiment was repeated several times with the same outcome and with a

maximum of 7.6% deviation in the cell-surface HLA class I antigen down-

regulation for each time point. (B) Relative levels of HLA A and HLA B. mRNA

from Jurkat cells that were not transfected (lane 1) or that were transfected with

backbone EGFP/Neo-diipMG plasmid (no U6shRNA, lane 2) or EGFP/Neo-

diipMG plasmid modified to express 6 copies of the scrambled shRNA (lane 3)

was used for RT-PCR using HLA A- and HLA B-specific primers and resolved by

agarose gel electrophoresis. Densitometry analysis revealed that the ratio of

HLA A replicon to HLA B replicon is approximately 3:1 (data not shown).




being evaluated in adoptive immunotherapy clinicaltrials.

The ability to disrupt antigen presentation by down-regulating HLA gene expression using RNAi is anapproach to avoiding T-cell-mediated immune recogni-tion, which might be used to facilitate transplantationand/or adoptive immunotherapy between HLA-divergentindividuals or to prolong the in vivo survival of trans-ferred T cells that express vector-encoded immunogenictransgenes and is a step toward the construction ofpremade buniversalQ T cells expressing tumor-specificchimeric immunoreceptors that dock with antigen inde-pendent of MHC, which could be readily available foradoptive immunotherapy of HLA-disparate recipients.

The conceptually straightforward plan described herefor introducing successive U6shRNA cassettes into anexpression vector should be generally useful for otherapplications in which controllable levels of RNAi-medi-ated target knockdown are desired. In those instances, ashRNA refractory site can be selected as target anddifferent levels of RNA down-regulation can be achievedby varying the number of U6shRNA cassettes. In addi-tion, the multimerization of the U6shRNA cassette is avaluable approach in the event that there are restrictionson the selection of the target site or when multiple genesneed to be simultaneously down-regulated. Hence, thistechnology should help address limitations to RNAi-induced gene silencing that depend on achievingadequate intracellular levels of siRNA [11]. Additionalchanges can be envisioned to improve further the efficacyof the siRNA vector, such as using enhancer elements,alternative Pol III promoters, and a combination ofsiRNAs directed to different regions of the selectedgene(s). Targeting different regions of the HLA genesand/or targeting multiple essential components of anti-gen processing and MHC (class I and II) expression usingthis strategy could further improve the simultaneousdown-regulation of HLA class I genes and completely

settes resulting in augmented down-regulation of classical HLA class I protein

ts were analyzed for 5 days to determine the RNAi kinetics represented by the

pression was limited to Jurkat cells that coexpressed EGFP to normalize for

of Jurkat cells transfected with EGFP/Neo-diipMG plasmid expressing 6 copies

and it is shown at day 6 (X). Maximal siRNA effect 4 days after transfection fits

repeated several times with the same outcome, and with a maximum of 6%

nd 6 located at the top indicate the number of copies of U6shRNA cassettes

A cassette results in augmented and durable down-regulation of classical HLA

iipMG plasmid with 0 to 6 copies of the active U6shRNA cassette or the 6-copy

f PE-conjugated anti-h2-m (x axis) and CyChrome-conjugated anti-HLA ABC

rface, on EGFP+ cells. The binding of isotype control mAbs is shown. The

r each plot. (C) Southern blot analysis demonstrating integration of plasmids

re transfected with up to 6 copies of the anti-HLA ABC U6shRNA cassette.

is of siRNA. Expression levels of shRNA in G418-resistant genetically modified

cific shRNA, probed using an oligonucleotide complementary to the antisense

enous U6 small nuclear (sn) RNA was used as an internal RNA loading standard.

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FIG. 4. (A) Identification of clone expressing siRNA with low levels of HLA A3. Binding of HLA A3-specific mAb and isotype control mAb to Jurkat T cell clone, 1A9,

transfected with the EGFP/Neo-diipMG plasmid modified to express 6 copies of the HLA ABC-specific shRNA cassette (6 copies U6 shRNA (clone 1A9)), and the

Jurkat cell line from which it was derived (6 copies U6shRNA) is shown. Columns 1 and 2 show transfected Jurkat cell lines expressing no shRNA (J.C. (0 copies))

or the scrambled version (6 copies U6shRNA scr). The level of HLA A3 cell-surface expression was measured by flow cytometry on EGFP+PI� cells using biotin-

conjugated mAb specific for HLA A3 and PE-conjugated streptavidin and is described by the median fluorescence intensity (MFI) F coefficient of variation. The

binding of an isotype control mAb demonstrates the minimal MFI (6 copies U6shRNA Isotype mAb). (B) Northern blot analysis of siRNA. SiRNA expression is

shown for Jurkat clone 1A9 (6 copies U6 shRNA (clone 1A9)), the Jurkat cell line expressing 6 copies of the shRNA cassette from which the clone was derived (6

copies U6 shRNA), and G418-resistant Jurkat cells transfected with 6 copies of the scrambled shRNA cassette (6 copies U6 shRNA scr). A probe complementary to

the endogenous U6 snRNA was used as a control for the integrity and amount of RNA analyzed in each lane. (C) HLA A3� cells transfected with HLA ABC-specific

siRNA are protected from T-cell-mediated specific lysis. The percentage of PI� EGFP+ HLA A3� target cells relative to PI� EGFP+ HLA A3+ cells was measured after

incubating RLRPGGKKK-pulsed 1A9 Jurkat clone with HIV-specific HLA A3-restricted T cell clone 28A2-15.


block T-cell-mediated immune recognition between HLA-divergent individuals.

MATERIALS AND METHODS

Construction of siRNA expression vectors. The plasmid HyTK-pMG was

generated from pMG (InvivoGen, San Diego, CA, USA) using site-directed

mutagenesis to remove a PacI restriction enzyme (RE) site at position 307

and replacing the Hy gene with the HyTk fusion gene, which combines

the hygromycin phosphotransferase gene with the herpes thymidine

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kinase (Tk) suicide gene [12,13]. The Neo-diipMG DNA vector was

modified from HyTK-pMG by replacing the hygromycin phosphotrans-

ferase (Hy) gene with the neomycin phosphotransferase (NeoR) gene and

removing intron A and the IRES element by BstXI and SpeI digestion. The

plasmid EGFP/Neo-diipMG contains the enhanced green fluorescent

protein gene (EGFP) blunt-end ligated into a unique NheI site under

control of the hybrid EF1a promoter. The 375-bp human U6 Pol III

promoter (�264 to +1) and small hairpin RNA cassettes were constructed

by PCR [14] with inclusion of a SalI RE site 5V of the promoter and

insertion of the shRNA sequences at position +1 of the U6 transcripts, and

the shRNA sequences are followed by the Pol III terminator signal



FIG. 5. Phenotypic effects of HLA A-specific siRNA in differentiated primary human T cells. Down-regulation of cell-surface HLA A2 (and HLA ABC, inset) protein

expression on hygromycin-resistant heterozygous (donor 1, HLA A*0201/0301, B*0702/1402) or homozygous (donor 2, HLA A*0201/0201, B*0702/3503) HLA

A2+ primary T cells transfected with a HyTK-pMG DNA plasmid modified to express 6 copies of the shRNA cassette. T cells were analyzed by flow cytometry for

binding of PE-conjugated anti-HLA A2 and HLA ABC. Dead cells were excluded by uptake of PI.


(TTTTTT) and two RE sites (XhoI and NotI) to form a U6shRNA cassette.

The cloned U6shRNA cassettes were validated by DNA sequencing. The

HLA ABC-specific shRNA sequence was 5V-GGAGATCACACTGACCTGG-

CAtttgtgtagTGCCAGGTCAGTGTGATCTCC-3V. The scrambled variant of

this shRNA sequence was 5V-GGAGATCACGTGTACCTGGCAtttgtg-

tagTGCCAGGTACACGTGATCTCC-3V. The HLA A-specific shRNA

sequence was 5V-CACCTGCCATGTGCAGCATGAtttgtgtagTCATGCTGCA-

CATGGCAGGTG-3V. To accommodate the requirement for an initiating G

in U6 transcripts, needed for efficient priming of transcription, an

additional guanine was included prior to the first 5Vnucleotide of the

HLA A-specific shRNA sequence [6]. One to six copies of the U6 promoter

and dsRNA (designated U6shRNA) cassettes, digested with SalI and NotI,

were directionally cloned into the unique XhoI and NotI sites of the EGFP/

Neo-diipMG or HyTK-pMG plasmids, and the number of inserted copies

was validated by sequencing and by agarose gel electrophoresis after

digestion with NotI and EcoRI.

Cells. Jurkat and primary T cells were maintained in T cell medium: RPMI

1640 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 2 mM

l-glutamine (Irvine Scientific), 25 mM Hepes (Irvine Scientific), 100 U/ml

penicillin, 0.1 mg/ml streptomycin (Irvine Scientific), and 10% heat-

inactivated defined fetal calf serum (FCS) (Hyclone, Logan, UT, USA).

Some Jurkat transfectants were cloned by limiting dilution in 96-well

plates after sorting for loss of class I HLA expression. Primary T cells were

expanded from peripheral blood mononuclear cells (PBMC) derived from

healthy volunteers using previously described methods [13]. Typically,

106 T cells were restimulated every 14 days by adding 30 ng/ml anti-CD3

(OKT3; Ortho Biotech, Raritan, NJ, USA), 50 � 106 irradiated PBMC, and

107 irradiated LCL. Recombinant human IL-2 (Chiron, Emeryville, CA,

USA) at 25 U/ml was added every 48 h, beginning on day 1 of culture.

Transfection. Transfection of Jurkat cells with 5 Ag of DNA plasmid,

linearized at the unique PacI RE site, was achieved by electroporating 100

Al of Jurkat cells at 30 � 106/ml in Nucleofector Solution V using a

Nucleofector I electroporator device operating under program T-14, per



the manufacturer’s conditions (Amaxa GmbH, Cologne, Germany). To

achieve stable transfections, a cytocidal concentration of G418 sulfate

(Calbiochem, La Jolla, CA, USA) of 1 mg/ml was added 72 h after

electroporation. Transfection of primary HLA A2+ human T cells in PBMC

was achieved 3 days after stimulation with 30 ng/ml OKT3 by electro-

porating with a single pulse of 250 V for 40 As 400 Al of 20 � 106/ml T cells

in hypo-osmolar buffer using a Multiporator device (Eppendorf AG,

Hamburg, Germany) with 5 Ag of linearized DNA plasmid [13]. Following

electroporation, cytocidal concentrations of G418 (0.8 mg/ml) or hygro-

mycin (0.2 mg/ml; InvivoGen) were added on day 5 of each 14-day

culture cycle.

Flow cytometry. The PE-conjugated and CyChrome-conjugated (clone

G46-2.6) mAbs specific for HLA ABC (BD Biosciences, San Jose, CA, USA)

were used at a dilution of 1:20. The biotin-conjugated mAbs specific for

HLA A3 and HLA B7/B27 (One Lambda Corp., Canoga Park, CA, USA)

were used at a dilution of 1:20. Staining of cells was accomplished in

Hanks’ balanced salt solution (Irvine Scientific) containing 2% FCS. In

some experiments, nonviable cells that had taken up 1 Ag/ml propidium

iodide (PI) were excluded from analysis. Data acquisition was performed

on a FACScan (BD Biosciences) and the percentage of cells in a region of

analysis, median fluorescence intensity, and coefficient of variation were

calculated using CellQuest version 3.3 (BD Biosciences). Fluorescence-

activated cell sorting, MoFlo MLS (Dako-Cytomation, Fort Collins, CO,

USA), was performed on Jurkat cells genetically modified with the EGFP/

Neo-diipMG plasmid expressing 6 copies of the U6 HLA ABC-specific

shRNA cassette.

Northern blotting. RNA was extracted from G418-resistant Jurkat cells

stably transfected with the various constructs expressing multimers of the

U6shRNA cassettes. The RNA was isolated using RNA STAT-60 (TEL-TEST

bBQ, Inc., Friendswood, TX, USA) according to the manufacturer’s

instructions. Fifteen micrograms of total RNA was fractionated in 8 M

6% PAGE and electroblotted for 2 h onto Hybond-N+ membrane

(Amersham Pharmacia Biotech). A 32P-radiolabeled 21-bp probe comple-

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mentary to the siRNA antisense strand was used for the hybridization

reactions, which were performed for 16 h at 378C. For size analysis, a 21-

mer DNA oligonucleotide was run alongside the RNA samples (not

shown). A 32P-radiolabeled 20-base probe complementary to the endog-

enous U6 snRNA was used as a control for the amount and integrity of the

RNAs analyzed in each lane.

Southern blotting. Ten micrograms of genomic DNA was digested

overnight with EcoRI and NotI RE, flanking the U6shRNA cassette(s).

The DNA was run on a 0.8% agarose gel and denatured by soaking for 50

min at room temperature in 1.5 M NaCl, 0.5 N NaOH. The gel was rinsed

with water and neutralized at room temperature in 200–300 ml of 1 M

Tris, pH 8, 1.5 M NaCl. The genomic DNA was transferred overnight by

capillary blotting with 10� SSC onto a Hybond-N+ (Amersham Pharmacia

Biotech) membrane. The membrane was UV cross-linked and prehybri-

dized in 50% formamide, 5� SSPE, 0.5% SDS, 5� Denhardt’s, and carrier

DNA (2.5–3.5 mg/50 ml), for 4 h at 428C. The probe for hybridization was

obtained by digesting the EGFP/Neo-diipMG + U6shRNA plasmid with

EcoRI and BamHI RE and labeling the resulting ~320-bp U6shRNA cassette

with the Random Primer Labeling system kit (Amersham Pharmacia

Biotech). Following overnight hybridization at 428C, the membrane was

washed once at 378C with 6� SSPE, 0.1% SDS for 10 min and twice with

2� SSPE, 0.1% SDS for 10 min.

RNA and cDNA preparation. Total RNA was extracted using the RNeasy

Mini Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufactur-

er’s instructions. cDNA was made by reverse transcription for 1 h at 428Cfrom 3 Ag of RNA, 67 pmol of oligo(dT), 0.2 mM dNTP, 0.3 Al of RNase

inhibitor, 0.1 M DTT, and 1 Al of reverse transcriptase (200 U, Superscript

II; Invitrogen, Carlsbad, CA, USA) in a 30-Al reaction. The reaction was

then heat inactivated at 958C for 5 min and the mixture was used directly

for PCR.

RT-PCR. To determine simultaneously the relative HLA-A and HLA-B

mRNA levels, first-strand cDNA from Jurkat cells was used to PCR-amplify

(1 cycle of 958C for 9 min; 20 cycles of 948C for 30 s, 588C for 20 s, 728Cfor 30 s; and 1 cycle of 728C for 10 min) both the 250-bp replicon using

HLA A-specific primers and a 150-bp replicon using HLA B-specific

primers, which were resolved by electrophoresis in a 1.5% agarose gel

developed with ethidium bromide (ETBr). The HLA A-, B-, and C-specific

5Vprimer was designated Q-ABC5 (5V-GCTGTGGTGGTGCCTTCTGG-3V),

the HLA A-specific 3Vprimer was designated A013 (5V-CCTGGGCACTGT-

CACTGCTT-3V), and the HLA B-specific 3Vprimer was designated B013

(5V-CCTGGGCACTGTCACTGCTT-3V) [15]. The relative binding of these

primers to an HLA class I sequence is shown (Fig. 1C). The PCR cycles were

predetermined to be in the linear range. Quantification was undertaken

using LabWorks Image Acquisition and Analysis software, version 4.0.0.8

(UVP BioImaging Systems, Upland, CA, USA) normalizing for the ETBr in

a 1-kb ladder (NEB; Beverly, MA, USA). Independent PCRs, normalizing

against GAPDH in each sample, ensured that the two sets of primers used

in each PCR did not compete with each other. Since the primers span an

intron, genomic contamination was excluded by the absence of high-

molecular-weight PCR product.

CTL-mediated lysis of HLA A3+ Jurkat cells. A Jurkat EGFP+ clone was

incubated in serum-free medium for 15 h at 378C with the HIV peptide

RLRPGGKKK [11] at 1 Ag/ml. This concentration of peptide resulted in

maximal CTL-mediated specific lysis of nontransfected HLA A3+ Jurkat

parental cells using a standard 4-h chromium release assay (CRA) (data

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not shown). After washing to remove unbound peptide, the RLRPGGKKK-

specific HLA A3+ CD8+ T cell clone 28A2-15 [9] was added at an

effector:target (E:T) ratio of 5:1 and the cells were cocultured for 4 h at

378C. This E:T ratio results in maximal lysis, assessed by CRA of

nontransfected Jurkat parental cells that have been incubated with 1

Ag/ml peptide (data not shown). The cells were then stained with biotin-

conjugated mAb specific for HLA A3, followed by PE-conjugated

streptavidin, and analyzed by flow cytometry for the presence of PE

staining on the remaining EGFP+ cells that excluded 1 Ag/ml PI.

ACKNOWLEDGMENTS

We thank Drs. Deborah and David Lewinsohn (Oregon Health and Science

University) for providing the HLA A3-restricted CTL clone, Dr. Dong Ho Kim

(Beckman Research Institute) for analyzing the h-IFN response, Dr. David

Senitzer (City of Hope) for HLA typing, Lucy Brown (City of Hope) for flow

cytometry sorting assistance, and Dr. Julie Mayo for editorial assistance. This

work was supported by CA30206, CA33572, HL74704, The Leukemia and

Lymphoma Society, The Lymphoma Research Foundation, The Pediatric Cancer

Research Foundation, and The Sidney Kimmel Foundation for Cancer Research.

RECEIVED FOR PUBLICATION NOVEMBER 29, 2004; ACCEPTED DECEMBER

27, 2004.

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Amplification of RNAi—Targeting HLA mRNAs

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