disruption of hla class ii antigen presentation in burkitt lymphoma: implication of a 47 000 mw...

Disruption of HLA class II antigen presentation in Burkitt

lymphoma: implication of a 47 000 MW acid labile protein in CD4+

T-cell recognition

Jason M. God,1 Dan Zhao,1 Chris-

tine A. Cameron,1 Shereen Amria,1

Jennifer R. Bethard2 and Azizul

Haque1

1Department of Microbiology and Immunol-

ogy, Hollings Cancer Center, and Children’s

Research Institute, Medical University of

South Carolina, Charleston, SC, and2Department of Cell and Molecular Pharma-

cology and Experimental Therapeutics, Medi-

cal University of South Carolina, Charleston,

SC, USA

doi:10.1111/imm.12281

Received 18 December 2013; revised 10

March 2014; accepted 11 March 2014.

Correspondence: Dr Azizul Haque, Depart-

ment of Microbiology and Immunology,

Medical University of South Carolina, 173

Ashley Avenue, BSB-201, Charleston, SC

29425, USA. Email: [email protected]

Senior author: Azizul Haque

Summary

While Burkitt lymphoma (BL) has a well-known defect in HLA class I-

mediated antigen presentation, the exact role of BL-associated HLA class

II in generating a poor CD4+ T-cell response remains unresolved. Here,

we found that BL cells are deficient in their ability to optimally stimu-

late CD4+ T cells via the HLA class II pathway. This defect in CD4+ T-

cell recognition was not associated with low levels of co-stimulatory

molecules on BL cells, as addition of external co-stimulation failed to

elicit CD4+ T-cell activation by BL. Further, the defect was not caused

by faulty antigen/class II interaction, because antigenic peptides bound

with measurable affinity to BL-associated class II molecules. Interest-

ingly, functional class II–peptide complexes were formed at acidic pH

5�5, which restored immune recognition. Acidic buffer (pH 5�5) eluate

from BL cells contained molecules that impaired class II-mediated anti-

gen presentation and CD4+ T-cell recognition. Biochemical analysis

showed that these molecules were greater than 30 000 molecular weight

in size, and proteinaceous in nature. In addition, BL was found to have

decreased expression of a 47 000 molecular weight enolase-like molecule

that enhances class II-mediated antigen presentation in B cells, macro-

phages and dendritic cells, but not in BL cells. These findings demon-

strate that BL likely has multiple defects in HLA class II-mediated

antigen presentation and immune recognition, which may be exploited

for future immunotherapies.

Keywords: antigen presentation; Burkitt lymphoma; enolase-like mole-

cules; HLA class II; immune escape.

Introduction

Burkitt lymphoma (BL) is a high-grade B-cell malignancy

and is one of the fastest growing malignancies in

humans.1–3 It occurs most frequently in children in areas

with holoendemic and hyperendemic malaria (endemic

BL) and is found with lower frequency in all other parts

of the world (sporadic BL), accounting for 1–2% of all

lymphomas in western countries.4–6 In addition to geo-

graphic distribution, BL may vary in its clinical manifes-

tation, with endemic BL presenting as tumours of the jaw

and sporadic BL causing the formation of tumours in the

gut and upper respiratory tract.1,7,8 In both adults and

children, BL is frequently associated with immune defi-

ciency.

A universal characteristic of all BL is the translocation of

the MYC gene to an immunoglobulin locus, which results

in its constitutive activation and over-expression.9–11 MYC

encodes the oncogenic transcription factor c-myc, and

studies indicate that up to 15% of all known genes may

lie in its target gene network.8,12,13 Another feature of BL

that is observed with varying frequencies depending on

Abbreviations: BL, Burkitt lymphoma; B-LCL, B-lymphoblastoid cell line; CII, type II collagen; CPB, citrate phosphate buffer;CTL, cytotoxic T lymphocyte; EBNA-1, Epstein–Barr virus nuclear antigen 1; EBV, Epstein–Barr virus; HLA, human leucocyteantigen; HSA, human serum albumin; IL-2, interleukin-2; MFI, mean fluorescence intensity; MW, molecular weight

ª 2014 John Wiley & Sons Ltd, Immunology, 142, 492–505492

IMMUNOLOGY OR IG INAL ART ICLE

the geographical location is association with Epstein–Barrvirus (EBV). EBV infection is seen in > 90% of endemic

BL but only 10–15% of sporadic BL, and has received

much attention as a possible co-factor for the develop-

ment of BL.4,6,8 EBV products are involved in the trans-

formation of BL cells and may also contribute to their

decreased immunogenicity. In addition to BL, EBV has

also been associated with other malignancies such as

Hodgkin’s lymphoma, transplant-related B-cell lympho-

mas, T-cell lymphomas, adult T-cell leukaemia, natural

killer leukaemia, and other lymphoid diseases.14–17 While

surgery, radiation, and chemotherapy have been used to

treat lymphoma patients, immunotherapy has shown

great promise with a positive long-term effect on overall

survival.18,19

Studies suggest that BL cells are deficient in their ability

to stimulate CD8+ T cells via the interaction of human

leucocyte antigen (HLA) class I with T-cell receptors.20–22

While the antigen-specific lysis of tumours is predomi-

nantly a function of HLA class I-restricted CD8+ T-cell

activation, HLA class II-restricted CD4+ T-cell function is

crucial in maintaining sustained immune responses to

tumours.23,24 Activation of natural killer cells has been

shown to induce long-term cytotoxic T lymphocyte (CTL)

memory, but CD4+ T cells remain essential for sustained

immune responses and complete destruction of tumours

by CTLs.25,26 The deficiency in the HLA class I-mediated

stimulation of CD8+ T cells by BL has been well studied

and stems from the poor immunogenicity of EBV nuclear

antigen 1 (EBNA-1).27–29 However, it remains unclear

why the immune system is unable to mount an effective

HLA class II-restricted response against BL. It has been

suggested that one EBV gene product, gp42, is involved in

blocking the HLA class II/T-cell receptor interaction.30

Gp42 is an EBV envelope protein that mediates virus

binding through its interaction with HLA class II, and

there is evidence that it may also impair CD4+ T-cell acti-

vation by blocking interaction of the T-cell receptor with

HLA class II on the B-cell surface.30–32 Previous research

in our laboratory has also suggested that B-cell lympho-

mas are deficient in presenting antigen via HLA class II

molecules.33 In the study presented here, we explore fur-

ther the nature of this defect, specifically in BL.

The expression of co-stimulatory molecules (CD80/86)

on antigen-presenting cells (APCs) is important to the

activation of CD4+ T cells, yet BL cells have a decreased

expression of these molecules.32 HLA class II antigen

presentation to T cells in the absence of co-stimulation

does not activate the T cell, but results in T-cell anergy.

With this in mind, we investigated whether the addition

of co-stimulatory signals, concomitant with HLA class II

antigen presentation, could help BL cells to stimulate

activation of CD4+ T cells.

Our current study suggests that there may be multiple

defects in BL cells resulting in a failure to stimulate CD4+

T cells. We demonstrate that BL cells and EBV-immortal-

ized B-lymphoblastoid cells (B-LCL) express similar

detectable levels of a transfected HLA class II allele and

that this HLA class II binds to exogenously delivered anti-

genic peptides to form class II–peptide complexes. How-

ever, in contrast to B-LCL, BL cells showed an impaired

capacity to activate CD4+ T cells. This deficiency in BL

was not overcome by the addition of external co-stimula-

tion. We also show that class II-mediated antigen presen-

tation capacity was restored in BL following incubation in

an acidic buffer (pH 5�5), and acid eluate obtained from

BL contained immunomodulatory molecules that

impaired HLA class II-mediated antigen presentation in

B-LCL. We further demonstrate the decreased expression

of an immunostimulatory, 47 000 molecular weight

(MW), enolase-like protein in BL which enhances antigen

presentation in B cells, macrophages and dendritic cells,

but not in BL. These findings suggest the presence of

multiple defects in HLA class II-mediated antigen presen-

tation in BL, which may provide novel targets for the

future development of immunotherapies against lym-

phoid malignancies.

Materials and methods

Cell lines

Human BL cell lines (Ramos, Ous) and an acute lym-

phoblastic leukaemia line (Nalm-6) were maintained in

complete RPMI-1640 supplemented with 10% fetal

bovine serum (Invitrogen, Carlsbad, CA), 50 U/ml peni-

cillin, 50 lg/ml streptomycin and 1% L-glutamine (Me-

diatech, Manassas, VA).33 Nalm-6 cells express cellular

markers and growth patterns characteristic of BL. The

6.16 cells are a bare lymphocyte syndrome-like mutant

line that is defective in a component of the regulatory

factor X (RFX) transcription couple. Briefly, the 6.16

line is a sub-clone of the parental 6.1.6 bare lymphocyte

syndrome-like line.34,35 The 6.16 line was transfected

with DR4 to generate the 6.16.DR4 line, and was further

transfected with DM to generate the 6.16.DR4.DM

line.36 The 6.16.DR4.DM line expresses DR, DM and Ii

molecules.37 The 6.16.DR4.DM line and a wild-type

human B-lymphoblastoid cell line (Frev) were main-

tained in Iscove’s modified Dulbecco’s medium supple-

mented with 10% bovine growth serum (Hyclone,

Logan, UT), 50 U/ml penicillin, 50 lg/ml streptomycin

and 1% L-glutamine (Mediatech). The wild-type BL line

Ous, and the wild type B-LCL line Frev constitutively

express HLA-DR4 molecules. Nalm-6 and Ramos cells

were retrovirally transfected for constitutive expression

of HLA-DR4 (DRB1*0401) with linked drug selection

markers for hygromycin and histidinol resistance to gen-

erate Nalm-6.DR4 and Ramos.DR4.36,38,39 HLA-DR4-

expressing dendritic cells (FSDC.DR4) and monocytic

ª 2014 John Wiley & Sons Ltd, Immunology, 142, 492–505 493

Defects in CD4+ T-cell recognition of BL

macrophages (THP-1.DR4.DM) were also generated as

described previously.36,38,39 Surface HLA-DR4 expression

was confirmed by flow cytometric analysis using the

DR4-specific monoclonal antibody, 359-F10.36,40,41 T-cell

hybridomas 2.18a and 1.21 were generated by immuni-

zation of DR4-transgenic mice as described elsewhere39,42

and respond to IgG j residues 188–203 and 145–159,respectively. T-cell hybridomas 4027/99 and 0921/98

(generously provided by Dr. Lars Fugger, Aarhus Uni-

versity Hospital, Aarhus, Denmark) are specific for DR4

and the immunodominant collagen peptide CII261–273.

The T-cell hybridoma 17.9 responds to human serum

albumin (HSA) residue 64–76K.43 T-cell hybridomas

were cultured in RPMI-1640 with 10% fetal bovine

serum, 50 U/ml penicillin, 50 lg/ml streptomycin, and

50 lM b-mercaptoethanol (Invitrogen).

Western blot analysis

Cell lysates obtained from Frev, Nalm-6.DR4,

Ramos.DR4, 6.16.DR4, 6.16.DR4.DM cell lines were sepa-

rated on 4% to 12% Bis/Tris NuPage gels in MES buffer

(Novex/Invitrogen), and analysed by Western blotting for

HLA-DM (Santa Cruz Biotechnology, Santa Cruz, CA)

protein expression as described.37,44 b-Actin was used a

loading control.33

Antigens and peptides

Human IgG j and cartilage bovine collagen type II (bCII)

were purchased from Sigma (St Louis, MO). The human

IgG immunodominant peptide j188–203 (sequence:

KHKVYACEVTHQGLSS), subdominant peptide j145–159(sequence: KVQWKVDNALQSGNS), type II collagen

peptide CII261–273 (sequence: AGFKGEQGPKGEP), and

human serum albumin peptide HSA64–76K (sequence:

VKLVNEVTEFAKTK) were produced using Fmoc tech-

nology and an Applied Biosystems Synthesizer (Applied

Biosystems, Foster City, CA) as described previ-

ously.39,42,43,45 The j188–203 and j145–159 peptides were

labelled as indicated at the a N-termini by the sequential

addition of two molecules of Fmoc-6-aminohexanoic acid

followed by a single biotin to yield the sequence biotin-

aminohexanoic acid–aminohexanoic acid–peptide. Mass

spectrometric analysis confirmed that the peptide was

tagged with a single biotin molecule at the N-terminus.

Peptide purity (> 99%) and sequence were analysed by

reverse-phase HPLC purification and mass spectroscopy.

Peptides were dissolved in PBS and stored at �20° until

used.

Antigen presentation assays

B-lymphoblastoid cells and BL cells were incubated with

whole IgG j antigen, j synthetic peptide, whole CII

antigen (40 lg/ml), CII synthetic peptide, or HSA peptide

(0–20 lM) for 3–24 hr at 37° in the appropriate cell cul-

ture medium.36,39 Cells were then washed and co-cultured

with the appropriate peptide-specific T-cell hybridoma

for 24 hr at 37°. In parallel assays, T-cell hybridomas

were stimulated with anti-CD3/CD28 before co-culture

with BL or B-LCL that had been incubated with j188–203or j145–159.

43 Following co-culture, T-cell production of

interleukin-2 (IL-2) was quantified by ELISA. Assays were

repeated in triplicate with standard error for triplicate

samples within a single experiment being reported. Addi-

tionally, 6.16.DR4.DM, Nalm-6.DR4, and Ramos.DR4

cells were washed once in citrate phosphate buffer (CPB;

pH 7�4 and pH 5�5), and incubated with the biotinylated

j peptides (b-j188–203 or b-j145–159) (5–10 lM) in the

respective CPB buffer for 4 hr. After incubation, cells

were washed twice with complete RPMI-1640 medium,

and co-cultured with the b-j188–203 or b-j145–159 peptide-

specific T-cell hybridomas (2.18a for j188–203, 1.21 for

j145–159) for 24 hr. T-cell production of IL-2 in the cell-

free culture supernatant was measured by ELISA as

described above.39 In separate T-cell assays, 6.16.DR4.DM,

Nalm-6.DR4, THP-1.DR4, and FSDC.DR4 cells were pre-

incubated with a 47 000 MW protein extract for 30 min,

followed by the addition of HSA64–76K peptide (5–10 lM)for 4 hr. Cells were washed twice in complete RPMI-1640

and co-cultured with the HSA64–76K peptide-specific T-cell

hybridoma (17�9) for 24 hr. Following incubation, T-cell

production of IL-2 in the cell-free culture supernatant was

measured by ELISA. Data are expressed as the mean �SEM of triplicate wells.

ELISA and ELISArray

Interleukin-2 (IL-2) levels in antigen presentation assay

supernatant were quantified by ELISA.46 A 96-well ELISA

plate was coated overnight at 4° with purified rat anti-

mouse IL-2 (Sigma). The plate was then washed and

blocked with 2% BSA at room temperature for 30 min.

After washing, standards and samples were plated in

appropriate wells and incubated at room temperature for

2 hr. A standard curve was generated using recombinant

IL-2 purchased from R&D (Minneapolis, MN). The plate

was washed, and biotinylated rat anti-mouse IL-2 (Sigma)

was added and incubated at room temperature for 1 hr.

Following washing, avidin peroxidase (Pierce, Rockford,

IL) was added to each well and incubated at room tem-

perature for 30 min. The plate was washed and p-nitro-

phenyl phosphate, disodium salt (PNPP) substrate

(Thermo Scientific, Rockford, IL) was added to each well

and incubated at room temperature. Readings were taken

every 30 min at 405 nm. Interleukin-2 levels in sample

wells are expressed in pg/ml, calculated from the standard

curve. Cytokines IL-4, IL-5, IL-10 and transforming

growth factor-b (TGF-b) in the cell-free culture


J. M. God et al.

supernatants were analysed by commercially available EL-

ISAarray kits (SABiosciences, Frederick, MD). Assays were

repeated in triplicate, and data were expressed as mean

pg/ml � SEM.

RT-PCR

Semi-quantitative PCR was performed on Nalm-6.DR4,

Ramos.DR4 and Frev cell lines for testing gene expression

of HLA-DR, HLA-DM and HLA-DO following total RNA

extraction using an RNeasy Mini Kit (Qiagen, Hilden,

Germany) according to the manufacturer’s protocol. A

reverse transcription reaction was performed using the

iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA).

b-actin mRNA was used as a control for each experiment

using the primer sequence 50-GACAGGATGCAGAAGGAGATTACT-30 (sense) and 50-TGATCCACATCTGCTGGAAGGT-30 (anti-sense). Primer sequences were also

used to test for HLA-DMb 50-GTCATGCCTCACAGCAGTGC-30 (sense) and 50-GGCTCAGGAGCCCCAA-30

(anti-sense); HLA-DOb 50- ATGTCCACTGGCCCTAT-

CAG-30 (sense) and 50-GCCACTCAGCATCTTTCTCC-30

(anti-sense); and Ii 50-GCTGTCGGGAAGATCAGAAG-30

(sense) and 50-GCCATACTTGGTGGCATTCT-30 (anti-

sense) gene expression. Thermal cycling parameters were

94° for 3 min, followed by 40 cycles of amplifications at

94° for 30 seconds, 60° for 1 min, 72° for 1 min and 72°for 5 min as the final elongation step. PCR products were

subjected to electrophoresis by using 1�5% agarose gel

and were visualized by ethidium bromide.

Peptide binding assays

Paraformaldehyde-fixed 6.16.DR4.DM, Nalm-6.DR4 and

Ramos.DR4 cells were incubated overnight with biotinylat-

ed jI or jII peptides in 150 mM CPB (pH 5�5 and 7�4),washed with PBS, and lysed on ice for 20 min with 50 mM

Tris buffer (pH 8) containing 0�15 M NaCl and 0�5% IGE-

PAL CA 630 (Sigma) as described previously.47,48 Cell su-

pernatants were added to plates (Costar, Cambridge, MA)

previously coated overnight with the anti-human class II

antibody 37.1 (kindly provided by L. Wicker, Merck

Research Laboratory, Rahway, NJ). The captured class II–peptide complexes were detected with europium-labelled

streptavidin (Pharmacia Fine Chemicals, Piscataway, NJ)

using a fluorescence plate reader (Delfia, Wallac, Turku,

Finland). The number of total DR molecules within B-

LCL/BL cells was quantified as described.39,42

Flow cytometric analysis

B-lymphoblastoid cells and BL cells were stained with

monoclonal antibodies directed against DR (L243) and

DR4 (359-F10), followed by secondary antibody labelled

with FITC. Cells treated with neutral (CPB, pH 7�4) and

acidic (CPB, pH 5�5) buffers as described in the binding

assays were also washed and stained with L243, followed

by secondary antibody labelled with FITC for cell surface

class II molecules. Samples were analysed on a FACScan

using CELLQUEST software (BD Bioscience, Mountain

View, CA). Background fluorescence was evaluated using

irrelevant isotype-matched monoclonal antibodies NN4

and IN-1 as described elsewhere.36,41,46

Protein extraction and digestion

Acid eluate was obtained from Nalm-6.DR4 and

6.16.DR4.DM as described previously.49 Extracts were

passed through a 30 000 MW filter, concentrated, and

protein concentrations were measured. Samples were

then run on a non-reducing gel and stained with Coo-

massie blue.37 Gel plugs were excised and placed in an

Eppendorf tube. Each plug was washed with 50 mM

ammonium bicarbonate for 10 min. Next, the plugs were

de-stained using 25 mM ammonium bicarbonate in 50%

acetonitrile for 15 min, repeated twice. The plugs were

dehydrated with 100% acetonitrile for 15 min, and dried

in a speedvac. Each gel plug was covered with Proteo-

mics Grade Trypsin (Sigma) and incubated at 37° over-

night. The supernatant was collected in a clean dry

Eppendorf tube. Peptides were further extracted with

one wash of 25 mM ammonium bicarbonate for 20 min

and three washes of 5% formic acid and 50% acetoni-

trile for 20 min each. The supernatant was collected and

pooled after each wash then dried down in a speedvac

to ~ 1 ll. Before analysis, the samples were reconstituted

with 10 ll of 0�1% trifluoroacetic acid. Samples were

then concentrated with a C18 Ziptip (Millipore, Billerica,

MA) and eluted with 0�1% trifluoroacetic acid, 50% ace-

tonitrile, and 7�0 mg/ml a-cyano-4-hydroxycinnamic acid

directly onto the matrix-assisted laser desorption/ioniza-

tion target.

Mass spectrometric analysis matrix-assisted laser desorp-tion/ionization time of flight/time of flight)

After the spots were dried completely, the plate was

loaded into the Applied Biosystems 4800 Proteomics

Analyzer.37 An external calibration was performed before

analysing samples, using the manufacturer’s standards

and protocols. Samples were analysed in batch mode

using 2000 laser shots per spectrum. First, peptide mass

maps were acquired over the m/z range of 800–3500 in

reflectron mode with a delayed extraction time opti-

mized for m/z 2000 by averaging 2000 scans to locate

peaks of peptide origin. The next batch run performed

mass spectrometry (MS)-MS analyses to obtain sequence

data on the 20 most abundant peaks from the MS analy-

sis. Upon completion of the batch processing, the data

were exported into the GPS EXPLORER data processing



system for interpretation and identification. The MAS-

COT database-searching algorithm analysed the data,

and summarized the results in report format. Database

searches were performed using two missed cleavages and

one differential modification of methionine oxidation.

The top 20 matches were reviewed before assigning con-

fident protein identifications. Proteins in acid eluate

preparations were also separated on large, non-reducing

10% polyacrylamide gels. A ~ 50 000 MW band was

excised from these gels, and the protein was extracted by

sonication in PBS on ice. The resulting extract was

added to BL cells or B-LCL and incubated with HSA

peptide for use in antigen presentation assays as

described.

Statistical analysis

The data are expressed as the mean (� SD) and analysed

using Student’s t-test or one-way analysis of variance,

with P ≤ 0�05 considered statistically significant.

Results

Diminished CD4+ T-cell response to HLA class II-mediated antigen presentation by BL cells

Burkitt lymphoma cells and B-LCL each express measur-

able levels of surface HLA class II molecules. However, to

gain a more direct comparison of class II-mediated anti-

gen presentation between these cell types, we expressed a

common HLA class II allele in several BL and B-LCL cell

lines. BL (Nalm-6 and Ramos) and B-LCL (6.16) were

retrovirally transfected to express the DR4 allele, HLA

DRB1*0401. Flow cytometric analysis showed that all

three cell lines were successfully transfected and constitu-

tively expressing the common DR4 allele (Fig. 1a).

6.16.DR4 cells were also transfected with HLA-DM to

generate 6.16.DR4.DM cells that express similar levels of

DM molecules when compared with Nalm-6.DR4 and Ra-

mos.DR4 cells (Fig. 1b). HLA-DR4-expressing BL (Nalm-

6.DR4 and Ramos.DR4) and B-LCL (6.16.DR4.DM) were

Figure 1. Burkitt lymphoma (BL) and BL type cells are deficient in HLA class II-mediated antigen presentation and CD4+ T-cell recognition.

(a) BL type cells (Nalm-6), BL (Ramos), and the B-lymphoblastoid cells (B-LCL) (6.16.DM) were retrovirally transfected to constitutively

express a common allele of HLA-DR4 (DR4B*0401). Cells were stained with a pan-HLA-class II antibody (L-243, dotted line) and an HLA-

DR4-specific antibody (359-F10, solid line). FACS plots are shown after retroviral transfection. Data are representative of at least three separate

experiments. (b) Western blot analysis for HLA-DM proteins in Nalm-6.DR4, Ramos.DR4, and 6.16.DR4.DM cells. (c) Nalm-6.DR4,

Ramos.DR4, and 6.16.DR4.DM cell lines were incubated with the synthetic j145–159 peptide (0–20 lM) for 4 hr, washed, and co-cultured with

the j peptide-specific T-cell hybridoma (1.21) for 24 hr. (d) Cells were incubated with the synthetic j188–203 peptide (20 lM) for 4 hr, washed

and co-cultured with the peptide-specific T-cell hybridoma (2.18a) for 24 hr. (e) Cells were also incubated with the whole antigen IgG j for

overnight, washed, and co-cultured with the j epitopes-specific T-cell hybridomas (2.18a for j188–203, 1.21 for j145–159) for 24 hr. Following

incubation, T-cell production of IL-2 in the cell-free culture supernatant was measured by ELISA. Data are expressed as the mean � SEM of

triplicate experiments.


J. M. God et al.

then sorted, matched for surface DR4 expression and

incubated in culture media with IgG j synthetic epitopes

(peptides j145–159 and j188–203) or whole antigen IgG.

Cells were then washed and co-cultured with the appro-

priate peptide-specific T-cell hybridoma (1.21 for j145–159and 2.18a for j188–203) for 24 hr. Interleukin-2 in the

resulting supernatant was measured by ELISA as

described.46 Data obtained from these assays revealed that

the B-LCL 6.16.DR4.DM efficiently and dose-dependently

presented the j145–159 peptide to stimulate T cells

(Fig. 1c). The 6.16.DR4.DM cells also functionally pre-

sented another synthetic peptide j188–203 to CD4+ T cells

(Fig. 1d). Although 6.16.DR4.DM cells elicited a strong

CD4+ T-cell response when presenting both synthetic jpeptides, Nalm-6.DR4 and Ramos.DR4 cells were unable

to stimulate the T-cell response (Fig. 1c,d). To examine

whether BL cells are also unable to process and generate

functional epitopes from the antigen IgG, we incubated

cells with the whole antigen overnight and tested for

T-cell responses. Again, 6.16.DR4.DM cells processed

whole IgG and presented both class II-restricted IgG jepitopes (j188–203 and j145–159) to T cells, whereas BL

cells (Nalm-6.DR4 and Ramos.DR4) were deficient in

their ability to stimulate CD4+ T cells with the same epi-

topes (Fig. 1e).

To examine whether wild-type BL cells constitutively

expressing the same class II allele can functionally deliver

j145–159 epitopes/peptides to T cells, we employed a wild-

type BL line Ous and a wild-type control B-cell line Frev.

Interestingly, Frev cells induced strong T-cell responses

and IL-2 production whereas Ous cells were unable to

stimulate the same T cells (Fig. 2a,b). To evaluate if the

same pattern is also true for other antigens, antigen pre-

sentation assays were carried out as described above, but

using a whole CII antigen or its synthetic version of an

epitope (CII261–273 peptide). As observed for whole IgG

and the synthetic IgG j peptides, 6.16.DR4.DM stimu-

lated a strong CD4+ T-cell response while Nalm-6.DR4

and Ramos.DR4 failed to elicit significant T-cell activa-

tion (Fig. 2c,d). Peptide presentation can be modulated

Figure 2. Disruption of class II–antigen presentation and CD4+ T-cell recognition of Burkitt lymphoma (BL). (a) A wild-type B-lymphoblastoid

cell line (B-LCL) (Frev) and a wild-type BL line (Ous), which constitutively express HLA-DR4 (DR4B*0401) allele, were incubated with the

whole antigen IgG j (0–20 lM) for overnight. (b) Cells were also incubated with the synthetic j145–159 peptide (0–20 lM) for 4 hr. After incuba-

tion, cells were washed and co-cultured with the j145–159-specific T-cell hybridoma line (1.21) for 24 hr. (c) Cells were incubated with another

self antigen type II collagen (CII; whole CII, 40 lg/ml) for overnight. (d) Cells were also incubated with the synthetic CII261–273 peptide (20 lM)for 4 hr. Cells were then washed and co-cultured with the CII261–273-specific T-cell hybridoma line 4027/99 for 24 hr. The production of IL-2 in

the cell-free culture supernatant was quantified by ELISA. (e) Western blot analysis for HLA-DM proteins in 6.16.DR4, 6.16.DR4.DM, and Frev

cells. (f) 6.16.DR4, 6.16.DR4.DM and Frev cells were incubated with the synthetic j145–159 peptide (20 lM) for 4 hr. Cells were washed and

co-cultured with the j145–159-specific T-cell hybridoma line (1.21) for 24 hr. The T-cell production of IL-2 was quantified as described. Data are

representative of the mean � SEM of triplicate experiments.



by HLA-DM molecules. To exclude the role of DM in

differential antigen presentation, we tested DM-expressing

6.16.DR4.DM and Frev cells as well as DM-negative

6.16.DR4 cells for their ability to present antigen to T

cells (Fig. 2e). All three cell lines efficiently presented the

j145–159 peptide to stimulate T cells regardless of DM

expression. Taken together, these data suggest that a

defect in HLA class II presentation is associated with BL

cells.

Addition of external co-stimulation fails to overcomediminished HLA class II-mediated antigenpresentation in BL cells although they possess class IIcomponents and produce only negligible amounts ofinhibitory cytokines

To examine whether B-LCL and BL cells express major

components of the HLA class II pathway, we performed

RT-PCR using Nalm-6.DR4 and Ramos.DR4 cells. A

wild-type B-cell line Frev, which expresses all the compo-

nents of the class II pathway, was used as a control. Data

showed that both B-LCL and BL lines expressed similar

levels of DM, DO, and Ii molecules (Fig. 3a). It is known

that BL cells express lower levels of co-stimulatory mole-

cules (CD 80/86) than B-LCL. To determine if the dimin-

ished CD4+ T-cell response to class II-mediated antigen

presentation by BL cells was a result of this deficiency,

antigen presentation assays were carried out as described,

but using CD4+ T cells that had been provided with

external co-stimulation. CD4+ T cells were stimulated

with IgG cross-linked anti-CD28 before co-culture with

BL cells or B-LCL presenting either j145–159 or j188–203synthetic peptide. The results shown in Fig. 3(b) demon-

strate that addition of external co-stimulation has little to

no effect on the ability of BL cells to present antigen via

HLA class II molecules. The B-LCL, 6.16.DR4.DM,

induced similarly high levels of IL-2 production, both in

the presence and absence of external co-stimulation, while

Figure 3. Analysis of major class II pathway components, requirement of co-stimulation and production of inhibitory cytokines in Burkitt lym-

phoma (BL)/B-lymphoblastoid cells (B-LCL). (a) RT-PCR analyses of Nalm-6.DR4, Ramos.DR4, and Frev cells for HLA-DM, HLA-DO, and Ii

gene expression. b-Actin was used as a loading control. Data are representative of at least three separate experiments. (b) Co-stimulation does

not overcome the BL defect in HLA class II antigen presentation. Nalm-6.DR4, Ramos.DR4, and 6.16.DR4.DM cells expressing a common allele

of HLA-DR4 (DR4B*0401) were incubated with synthetic j peptides (j188–203 or j145–159), washed, and co-cultured with the appropriate j pep-

tide-specific T-cell hybridomas (2.18a for j188–203, 1.21 for j145–159) with or without external co-stimulation by IgG cross-linked anti-CD28. Fol-

lowing incubation, T-cell production of IL-2 in the cell-free culture supernatant was measured by ELISA. (c) Cell supernatants obtained from

Nalm-6.DR4, Ramos.DR4, 6.16.DR4.DM and Frev cells, were analysed for IL-4, IL-5, IL-10, and TGF-b cytokines by ELISArray. Data are

expressed as the mean � SEM of triplicate wells.


J. M. God et al.

Nalm-6.DR4 and Ramos.DR4 again failed to stimulate a

CD4+ T-cell response under either condition. Analysis of

BL/B-LCL culture supernatants by ELISArray detected

negligible amounts of IL-4, IL-5, IL-10, and TGF-b(Fig. 3c), suggesting that these inhibitory cytokines were

not responsible for the observed defect in antigen presen-

tation by BL.

Antigenic peptides bind to BL-expressed HLA class IImolecules, and antigen presentation is restored in BLat acidic pH

To further assess the defect in HLA class II-mediated

antigen presentation by BL cells, we evaluated the binding

efficiency of antigenic peptides to HLA class II molecules

expressed on the surface of BL cells and B-LCL. Nalm-

6.DR4, Ramos.DR4, and 6.16.DR4.DM cells were fixed

with 1% paraformaldehyde, washed and incubated with

biotin-labelled synthetic j peptides (b-j145–159 or b-j188–203) in neutral (pH 7�4) or acidic (pH 5�5) CPB for 4 hr.

Cells were washed, lysed, and total class II–peptide com-

plexes were captured by ELISA, followed by detection

with europium-labelled streptavidin as described in the

Materials and methods. Data obtained revealed that

under neutral conditions, BL cells and B-LCL each bound

measurable levels of b-j145–159 or b-j188–203 peptides

(Fig. 4a). It was noted that the peptide binding to class II

proteins was much better at acidic pH than the neutral

pH (Fig. 4b), and the binding capacity was almost com-

parable for both BL cells and B-LCL. These data suggest

that the impaired class II presentation by BL cells is not

associated with a defect in peptide binding.

Having established that BL-expressed HLA class II pro-

tein is capable of binding antigenic peptides at pH 5�5with a higher affinity, we sought to determine if this

enhanced binding resulted in the formation of functional

class II–peptide complexes. Antigen presentation assays

were carried out using b-j188––203 and b-j145–159 peptides.Unlike regular antigen presentation assays, the initial

incubation of peptide with BL cells or B-LCL was carried

out under neutral and acidic pH conditions (7�4 and 5�5,respectively) to follow conditions used in binding assays.

Briefly, cells were incubated with biotin-labelled synthetic

j peptides (b-j188–203 or b-j145–159) in neutral (pH 7�4)or acidic (pH 5�5) CPB for 4 hr. Cells were then washed

and co-cultured with peptide-specific T cells in culture

media for 24 hr. Analysis of IL-2 in the culture superna-

tants of these assays showed that when peptide binding

Figure 4. Antigenic peptides bind to Burkitt lymphoma (BL) cell surface-expressed HLA class II proteins at both neutral and acidic conditions.

DR4+ BL (Nalm-6.DR4 and Ramos.DR4) and B-lymphoblastoid cells (B-LCL) (6.16.DR4.DM) were incubated with biotin-labelled j peptides

(j188–203 or j145–159) in citrate phosphate buffer (CPB) at pH 7�4 (a) and pH 5�5 (b) for 4 hr. Cell lysate was collected and class II–peptide com-

plexes were captured with anti-human HLA class II antibodies. Captured complexes were detected with europium-labelled streptavadin. Data are

the mean fluorescence � SEM of at least three separate experiments. (c,d) Nalm-6.DR4, Ramos.DR4 and 6.16.DR4.DM cells were also washed

once in CPB (pH 7�4 and pH 5�5), and incubated with biotinylated j peptides (b-j188–203 or b-j145–159) in CPB at pH 7�4 or pH 5�5 for 4 hr.

Cells were washed twice with complete RPMI-1640 medium, and co-cultured with the appropriate j peptide-specific T-cell hybridomas (2.18a

for j188–203, 1.21 for j145–159) for 24 hr. Following incubation, T-cell production of IL-2 in the cell-free supernatant was measured by ELISA.

Data are expressed as the mean � SEM of triplicate experiments.



was carried out at pH 7�4, antigen presentation by BL

cells and B-LCL mirrored the results from previous

assays, with Nalm-6.DR4 and Ramos.DR4 stimulating lit-

tle to no IL-2 production, while 6.16.DR4.DM elicited a

strong CD4+ T-cell response (Fig. 4c). However, when

peptide binding was carried out at pH 5�5, antigen pre-

sentation by Nalm-6.DR4 and Ramos.DR4 was restored

to levels nearly identical to 6.16.DR4.DM (Fig. 3d). These

data indicated that functional class II–peptide complexes

could be formed at low pH on BL cells, resulting in

CD4+ T-cell stimulation.

BL-derived molecules disrupt HLA class II antigenpresentation in B-LCL

To investigate whether incubation of cells in neutral and

acidic conditions altered cell surface class II molecules,

we stained Nalm-6.DR4 and 6.16.DR4.DM cells with a

pan class II antibody L243 for surface class II proteins

(Fig. 5a,b). Apparently, both cell lines expressed similar

levels of class II molecules in neutral [mean fluorescence

intensity (MFI) = 557�3 versus MFI = 568�1] and acidic

(MFI = 385�8 versus MFI = 379�5) conditions as analysedby flow cytometry. Results from antigen presentation

assays also suggested that at pH 5�5 molecules that inhibit

HLA class II-mediated antigen presentation are eluted

from the surface of BL cells, hence restoring the antigen-

presenting capacity of these cells. Consequently, we

sought to determine if the acid eluate obtained from BL

cells contained HLA class II inhibitory activity. Acid elu-

ate (pH 5�5) was collected from Nalm-6.DR4 and

6.16.DR4.DM and separated into < 30 000 and > 30 000

MW fractions. Antigen presentation assays were then con-

ducted with 6.16.DR4.DM being incubated with jI or jIIin the presence of the < 30 000 or > 30 000 MW frac-

tions from 6.16.DR4.DM or Nalm-6.DR4. Subsequent

steps of the antigen presentation and IL-2 ELISA quantifi-

cation were the same as already described. Data from

these experiments showed that the > 30 000 MW acid

eluate fraction from Nalm-6.DR4 greatly diminished the

capability of 6.16.DR4.DM to present j188–203 or b-j145–159 (Fig. 5c,d). The < 30 000 MW fraction had no effect.

To further characterize the inhibitory component in the

> 30 000 MW fraction, it was treated with proteinase K

or trypsin (data not shown) and used in antigen presen-

tation assays as above. Data shown in Fig. 5(e) revealed

that following proteinase K treatment, any inhibitory

activity associated with Nalm-6.DR4 acid eluate was

Figure 5. Burkitt lymphoma (BL) and B-lymphoblastoid cells (B-LCL) cells express comparable levels of class II proteins, but BL acid (pH 5�5)eluate contains HLA class II inhibitory molecules > 30 000 MW which are sensitive to proteinase K. (a) Nalm-6.DR4 (black line) and

6.16.DR4.DM (green line) cells were incubated in citrate phosphate buffer (CPB; pH 7�4), washed and stained with L-243 for surface class II mol-

ecules. (b) Cells were also incubated in CPB (pH 5�5), washed and stained for surface class II molecules. NN4 was used as an isotype control.

Acid elutions (pH 5�5) obtained from Nalm-6.DR4 and 6.16.DR4.DM were passed through 30 000 MW cut-off filters. Both the retained fraction

(> 30 000 MW) and the filtrate (< 30 000 MW) were collected. B-LCL cells (6.16.DR4.DM) were then incubated with j peptides (j188–203 or

j145–159) in acid eluates collected from Nalm-6.DR4 and 6.16.DR4.DM. Cells were washed and co-cultured with the appropriate j peptide-specific

T-cell hybridomas (2.18a for j188–203, 1.21 for j145–159). T-cell production of IL-2 in the cell-free culture supernatant was measured by ELISA.

(c) < 30 000 MW fraction, (d) > 30 000 MW fraction. Data are expressed as the mean � SEM of triplicate experiments. (e) The > 30 000 MW

fraction from 6.16.DR4.DM and Nalm-6.DR4 was then subjected to proteinase K treatment, dialysed and concentrated, and incubated with

6.16.DR4.DM and j peptides (j188–203 or j145–159). Cells were washed and co-cultured with the appropriate j peptide-specific T-cell hybridomas

as described. Following incubation, T-cell production of IL-2 in the cell-free culture supernatant was measured by ELISA. Data are expressed as

the mean � SEM of triplicate experiments. *P < 0�05.


J. M. God et al.

almost completely lost. These data suggest that BL-associ-

ated protein molecules impair HLA class II antigen pre-

sentation.

A 47 000 MW enolase-like molecule enhances HLAclass II antigen presentation in B-LCL, dendritic cells,and macrophages, but not in BL cells

Data collected in previous assays suggested the presence

of a proteinaceous HLA class II inhibitory component in

BL cell acid eluate, but not in B-LCL acid eluate. This led

us to compare protein expression patterns between the

two cell types at acidic pH. Acid eluate (pH 5�5) was col-lected from BL cells (Nalm-6.DR4) and B-LCL

(6.16.DR4.DM), and the proteins were separated on a

non-reducing gel, followed by staining with Coomassie

blue. Data from this assay showed a ~ 50 000 MW pro-

tein consistently expressed at high levels in 6.16.DR4.DM,

but seen only in low amounts in Nalm-6.DR4 (Fig. 6a).

A gel plug sample of this protein was cut and analysed by

mass spectrometry, revealing a 47 000 MW enolase-like

molecule (Accession number 4503571). Acid eluates from

a wild-type BL cell line (Ous) and B-LCL (Frev) also

showed a high level of the 47 000 MW molecule

Figure 6. Burkitt lymphoma (BL) and B-lymphoblastoid cells (B-LCL) differentially express an acid labile 47 000, enolase-like molecule, which

enhances HLA class II-mediated antigen presentation in B cells, macrophages and dendritic cells. Proteins in acid (pH 5�5) elutions from BL

(Nalm-6.DR4) and B-LCL (6.16.DR4.DM) and a wild-type B-cell line (Frev) were separated on a non-reducing gel. (a) Proteins were detected

and relative expression was determined by staining with Coomassie blue. Data shown are representative of at least three separate experiments. (b)

A band corresponding to ~ 50 000 MW was removed and analysed by matrix-assisted laser desorption/ionization time of flight/time of flight

mass spectrometry. Proteins in acid (pH 5�5) elution from 6.16.DR4.DM cells also were separated on a non-reducing gel. Following staining with

Coomassie blue, an approximately 50 000 MW band containing an enolase-like protein (~ 47 000 MW) was cut and the protein was extracted

by sonication in PBS on ice. The protein extract was added to HLA-DR4-expressing (c) BL (Nalm-6.DR4), (d) B-LCL (6.16.DR4.DM), (e) mac-

rophages (THP-1.DR4.DM) or (f) dendritic cells (FSDC.DR4) for 30 min, followed by the addition of HSA64–76K peptide for 4 hr. Cells were

washed and co-cultured with the HSA64–76K peptide-specific T-cell hybridoma (17.9) for 24 hr. Following incubation, T-cell production of IL-2

in the cell-free culture supernatant was measured by ELISA. Data are expressed as the mean � SEM of triplicate experiments. *P < 0�01,**P < 0�05.



consistently found in B-LCL, not in BL cells (data not

shown), suggesting that the enolase-like protein is being

suppressed in BL.

Results from protein expression pattern and mass spec-

tral analysis raised the possibility that, in addition to

expressing HLA class II inhibitory molecules, BL cells

may be deficient in expressing immune stimulatory pro-

teins. To further examine the role of the 47 000 MW

enolase-like molecule, which is highly expressed in B-LCL

but expressed at low levels in BL cells, proteins in acid

eluate (pH 5�5) samples from 6.16.DR4.DM cells were

separated on large, non-reducing gels. The band which

corresponded to our 47 000 MW enolase-like molecule

was excised and the protein was extracted by sonication

in PBS on ice. To confirm the presence of the 47 000

MW protein in the gel extract, samples were separated on

a non-reducing gel and stained with Coomassie blue.

Results confirmed that only the 47 000 MW protein was

present in the gel extract samples (Fig. 6b). Antigen pre-

sentation assays were then carried out with Nalm-6.DR4,

6.16.DR4.DM, dendritic cells (FSDC.DR4), and macro-

phages (THP-1.DR4.DM) being incubated with HSA64–

76K synthetic peptide in the presence of the 47 000 MW

enolase-like gel extract (Fig. 6c–f). Subsequent steps in

the antigen presentation assay and IL-2 quantification

were carried out as already described. Results from these

assays showed that the 47 000 MW enolase-like molecule

significantly enhanced antigen presentation by B cells,

macrophages, and dendritic cells, but not by the BL cells.

Discussion

HLA-mediated antigen presentation is central to the

immune system’s ability to detect and mount responses

to transformed cells. However, malignant B-cell tumours,

including BL, are equipped with strategies to escape

detection by the immune system.36,50 Studies have shown

that BL cells are not recognized as foreign or malignant

by CD8+ effector T cells that can directly kill transformed

cells. This defect has been well-characterized and results

from the poor immunogenicity of the EBNA-1 antigen,

which weakly stimulates CD8+ T cells.20,51 As EBNA-1 is

the lone EBV antigen synthesized in BL, few options are

available for CD8+ T-cell activation. In contrast, much

less is known about the role of HLA class II antigen pre-

sentation in the recognition of BL. While CD8+ T cells

are capable of directly killing tumour cells, it is now

understood that CD4+ T-cell activation is required for

lasting anti-tumour responses.52,53

Although BL cells express HLA class II proteins on

their cell surface and theoretically could prime CD4+ T

cells against their self-antigens, we observed that they

failed to do so. We found that BL-associated molecules

may block the signals that effectively trigger the CD4+ T-

cell activation. Specifically, we have shown that CD4+ T

cells failed to recognize antigens in association with HLA

class II molecules on BL cells, whereas B-LCL efficiently

processed and presented antigens to T cells in the context

of class II molecules. Second, BL cells incubated at pH

5�5 gained the ability to stimulate CD4+ T cells to pro-

duce IL-2 in an HLA class II specific manner. Third,

acid-eluted molecules associated with BL cells suppressed

B-LCL’s ability to present antigen to CD4+ T cells.

Fourth, BL/B-LCL cells produced extremely low levels of

inhibitory cytokines TGF-b, IL-10, IL-5, or IL-4; which

are unlikely to disrupt class II presentation. Finally, we

show that BL cells have decreased expression of a 47 000

MW enolase-like molecule, which enhances class II-medi-

ated antigen presentation in B cells, macrophages and

dendritic cells, but not in BL cells. These important find-

ings are directly applicable to other EBV-associated tumours

such as Hodgkin’s lymphoma, transplant-related B-cell lym-

phomas, and other malignant lymphoid diseases. Identifica-

tion of BL-associated inhibitory molecules (BLAIM) that

disrupt CD4+ T-cell recognition as well as deficiencies in

expression of immunostimulatory molecules will spur

future development of new therapies against B-cell lympho-

mas.

The expression levels of class II molecules, invariant

chain (Ii), and HLA-DM/DO molecules in APCs may

modulate antigen presentation to T cells.36,41,54 While

HLA-DM plays a critical role in the peptide selection pro-

cess, in the absence of DM editing, peptides may bind in

different conformations and are recognized by different

subsets of T cells.55–57 In this study, we did not observe

any significant differences in class II, Ii, and DM protein

expression in BL/B-LCL tested. Mutant B-LCL (6.16.DR4)

lacking DM molecules also efficiently presented class II-

restricted peptides to CD4+ T cells. In addition, accessory

proteins on APCs, such as co-stimulatory molecules, are

also necessary to optimally stimulate T cells. BL cells are

known to express lower levels of co-stimulatory molecules

than B-LCL,32 so it was possible that the defect we

observed in BL-mediated CD4+ T-cell stimulation

stemmed from this property. Defects in co-stimulation by

APCs can be overcome by adding antibodies to co-stimu-

latory receptors on T cells. For example, anti-CD28 anti-

bodies can serve as surrogates for the B7 co-stimulatory

molecule expressed on APCs. While our T-cell hybrido-

mas are not dependent on co-stimulatory molecules,39 we

still tested whether external co-stimulation could help BL

cells stimulate these T cells. Even in the presence of exter-

nal co-stimulation, these tumours failed to functionally

present antigens to CD4+ T cells. Other activating signals,

such as cross-linking IgM on BL cells, also failed to stim-

ulate CD4+ T cells (data not shown), indicating that

co-stimulation was not sufficient to overcome the defect.

Efficient binding of antigen to HLA is a requisite for

effective presentation to and activation of T cells. Having

established that our BL cell lines were deficient in their


J. M. God et al.

ability to stimulate the activation of CD4+ T cells, and

having determined that this deficiency was not the result

of a defect in co-stimulation, we considered the possibil-

ity of a defect in antigen binding to HLA-DR4. Data

from our binding assays suggest that peptides bind to

DR4 molecules on BL cells at both acidic and neutral

pH. Functional assays failed to detect CD4+ T-cell

responses to these class II–peptide complexes when

formed at neutral pH, suggesting a lack of CD4+ T-cell

recognition of BL. In contrast, class II–peptide complexes

formed on BL/B-LCL at acidic pH, were functional and

restored T-cell recognition to BL. Under these condi-

tions, BL/B-LCL also expressed similar levels of cell sur-

face class II proteins. This suggests that the lack of T-cell

recognition at neutral pH did not reflect a defect in pep-

tide binding to class II molecules on BL cells, but rather

a failure to form functional peptide–class II complexes

on these tumours.

We have shown further that acid eluates from B-LCL

cells did not influence synthetic j peptide presentation by

B-LCL, but those obtained from BL cells significantly

inhibited peptide presentation by B-LCL, suggesting that

BL-cell-derived acid-eluted molecules may disrupt HLA

class II-restricted T-cell recognition. Acid eluate obtained

from BL cells was passed through 30 000 MW cut-off fil-

ters and tested for inhibitory activity. Data showed that

BL-derived molecules retained in the 30 000 MW cut-off

filter (> 30 000 MW) significantly inhibited CD4+ T-cell

production of IL-2, suggesting that BLAIM could be

greater than 30 000 MW in size. Additionally, BLAIM

were sensitive to proteolytic enzymes as proteinase K

treatment negated their ability to block CD4+ T-cell rec-

ognition of BL cells. Treatment of samples with trypsin

also inhibited the activity of BLAIM (data not shown),

further implying that BLAIM may be proteinaceous in

nature. However, it remains possible that BLAIM could

be structure-dependent, domain-dependent, or even mod-

ified molecules.

Working under the premise that BLAIM may be pro-

teinaceous in nature, we then compared protein expres-

sion patterns between BL cells and B-LCL. While

initially looking for the over-expression of proteins in

BL that may be BLAIM, we consistently observed the

decreased expression of a ~ 50 000 MW protein in BL.

This led us to consider the possibility that, in addition

to expressing molecules that may inhibit immune stimu-

lation, BL cells may also be deficient in immunostimula-

tory molecules. Protein expression and mass

spectrometry analyses revealed that the predominant pro-

tein was a 47 000 MW enolase-like molecule. When this

protein was extracted from Coomassie-stained gels and

used in antigen presentation assays, it significantly

increased class II-mediated antigen presentation in mac-

rophages and dendritic cells as well as B-LCL, but not in

BL cells. Hence, in addition to expressing BLAIM, which

impairs CD4+ T-cell activation, BL cells express lower

levels of an immunostimulatory, 47 000 MW, enolase-

like molecule. Enolase-1 protein has been reported to be

a transcriptional repressor of c-myc, but can also func-

tion at the cell surface as a plasminogen receptor.58,59

The effect of our 47 000 MW extract on T cells was

tested, and no significant differences in cell proliferation

and IL-2 production were observed (data not shown).

Recently, a-enolase-1 has been considered as a marker of

pathological stress in a number of infectious and auto-

immune diseases such as inflammatory bowel disease,60,61

autoimmune hepatitis,62 and membranous glomerulone-

phritis.63

Future studies will focus on further characterizing

BLAIM and the 47 000 MW enolase-like molecule, as

well as elucidating their mechanisms of action. The suc-

cessful characterization of these molecules offers poten-

tial for the development of new immunotherapies to

more efficiently treat BL, potentially lessening the

dependence on harsh chemotherapeutic regimens.

Although currently used chemotherapeutic treatments

are successful, achieving high cure rates in both adults

and children, treatment-associated toxicities can be

severe and are not tolerated well by elderly or HIV-

infected patients.64 Use of the anti-CD20 monoclonal

antibody, rituximab, in conjunction with chemotherapy,

has led to increased response rates, but treatment-asso-

ciated toxicities remain problematic.65,66 Additionally,

the use of the immunosuppressive rituximab in patients

already immunocompromised with HIV infection is a

debated issue.66 When considered together, these issues

reveal the need for improvements in treatment options

for BL patients as well as patients with other lymphoid

malignancies. Exploiting BL’s over-expression of BLAIM

or decreased expression of the 47 000 MW enolase-like

protein provides the potential to generate a more tar-

geted immune response to these cells, possibly reducing

bystander effects and resultant treatment-associated tox-

icities.

Acknowledgements

We thank Dr Janice Blum (Indiana University School of

Medicine) for providing cell lines and reagents, and Dr

Daniel Knapp (Department of Pharmacology) for mass

spectrometry facility and technical assistance. We also

thank Bently Doonan for critical reading of the manu-

script. This work was supported by grants from the

National Institutes of Health (R01 CA129560 and R01

CA129560-S1) to A. Haque. The research presented in

this article was also supported in part by the Tissue Bior-

epository and Flow Cytometry Shared Resource as part of

the Hollings Cancer Center at the Medical University of

South Carolina, which is funded by a Cancer Center Sup-

port Grant P30 CA138313.



Disclosures

The authors have no financial conflict of interest.

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