Immunology 1999 96 114–121

Enhancing the immunotherapeutic potential of mycobacteria by transfection withtumour necrosis factor-a

J. L. HALEY,* D. G. YOUNG,* A. ALEXANDROFF,* K. JAMES* & A. M. JACKSON† *Department of Surgery, ListerSurgical Research Laboratories, University of Edinburgh, Royal Infirmary, Edinburgh, and †ICRF Cancer Medicine Research Unit,

St. James’ University Hospital, Leeds, UK

SUMMARY

In an attempt to enhance the anti-tumour properties of mycobacteria we have developedrecombinant forms of Mycobacterium smegmatis which express and secrete biologically activehuman tumour necrosis factor-a (TNF-a). This was achieved by transfecting M. smegmatis usingshuttle plasmids incorporating the cDNA sequence for the human TNF-a mature peptide. In vitroexperiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112)indicate that our genetically modified mycobacteria are more effective than wild-type at inducingor up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an arrayof proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte–macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokineexpression in response to mycobacteria transfected with vector containing no gene insert. However,this was not as pronounced as that observed following tumour cell stimulation by the TNF-a-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains werefound to down-regulate the secretion of the anti-inflammatory cytokine transforming growthfactor-b1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladdertumour cell growth and revealed that its antiproliferative potency is enhanced by transfectingwith human TNF-a and, to a lesser extent, with vector alone. All M. smegmatis strains wereeffective in the activation of peripheral blood leucocyte cultures. However, no differences wereobserved in the ability of the TNF-a-transfected, mock-transfected and wild-type mycobacteriato induce tumour cell killing activity. These results suggest that the immunomodulatory effects ofM. smegmatis can be enhanced by transfection with vectors which allow the secretion of humanTNF-a, thus increasing mycobacterial immunotherapeutic potential.

INTRODUCTION in almost 20% of CIS patients remain elusive. However, wedo know that the mycobacteria enter epithelial cells, probablyLive bacillus Calmette–Guerin (BCG), an attenuated strainby endocytosis5 and once inside the bladder wall, they initiateof Mycobacterium bovis, has been used as an immunothera-a large local influx of lymphocytes, macrophages, polymor-peutic agent against many forms of malignancy with varyingphonuclear cells and natural killer cells.6 It is believed thatdegrees of success.1–3 Despite its proven value in treatingthese cells give rise to the majority of cytokine moleculescarcinoma in situ (CIS) of the bladder,4 its mechanism ofdetected in the urine from patients, including interleukin-1action is not fully understood and reasons for therapy failure(IL-1), IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, tumour necrosisfactor-a (TNF-a) and interferon-c (IFN-c).7–12 However, it

Received 23 July 1998; revised 21 September 1998; acceptedhas recently emerged that cultured tumour cells themselves,24 September 1998.can be induced to secrete IL-1, IL-6, IL-8 and TNF-a in

Abbreviations: ADC, albumin dextrose complex; BAK, bacillus response to mycobacterial stimulation.13–15Calmette–Guerin (BCG)-activated killer (cell ); hsp, heat-shock pro- The release of cytokines during immunotherapy is believedtein; GM–CSF, granulocyte–macrophage colony-stimulating factor;

to be responsible for the marked changes in tumour cell surfaceICAM-1, intracellular adhesion molecule-1; LB, Luria–Bertaniphenotype observed following mycobacterial instillation,(medium); SMAK, Mycobacterium smegmatis-activated killer (cell );especially in major histocompatibility complex (MHC) classTCC, transitional cell carcinoma; TGF-b, transforming growth

factor-b. II and intracellular adhesion molecule-1 (ICAM-1)expression.16,17 It is thought that the induction or up-regulationCorrespondence: Dr J. L. Haley, Lister Research Laboratories,of these molecules may play a role in tumour recognition andDepartment of Surgery, University of Edinburgh, Royal Infirmary,

Lauriston Place, Edinburgh EH3 9YW, UK. destruction. For example, enhanced ICAM-1 expression is

© 1999 Blackwell Science Ltd114

Enhancing mycobacterial immunotherapeutic potential by genetic manipulation 115

believed to favour binding of lymphokine-activated killer (gfp).30 Recombinant gfp fluoresces in ultraviolet illuminationand is used as a selectable marker.(LAK) cells to tumour cells prior to killing,18–20 while MHC

class II expression on tumour cells has been associated withantigen presentation to CD4+ T cells21 propounding an Cloning of TNF-a

Digestion of the purified PCR product with appropriate restric-alternative specific immune response pathway.A number of workers have begun to employ recombinant tion enzymes (Promega UK Ltd, Southampton, UK) was

followed by ligation into the shuttle vectors and transformationtechnology for the purpose of elucidating the mechanismsbehind mycobacterial immunotherapy/vaccination.22–24 into competent DH5a cells (Gibco BRL, Life Technologies

Ltd, Paisley,UK). The transformants were selected on kana-Modifications to the immunostimulatory properties of myco-bacterial strains could provide clues to understanding the mycin–agar and incorporation of the gene insert was verified

by PCR using the original TNF-a primers. Successfully trans-difference between therapy success and failure and may ulti-mately provide a means of enhancing the clinical efficacy of formed E. coli colonies were grown up overnight in Luria–

Bertani (LB) liquid medium containing 30 mg/ml kanamycinBCG therapy.The aim of this study was to genetically modify (Gibco BRL) and the cultures were maxi-prepped (Maxi-kit

tip-500; Qiagen Ltd) to harvest the cloned vector constructs.Mycobacterium smegmatis, a live, non-pathogenic bacterium,to express human TNF-a. TNF-a was selected because of its After confirmation of the TNF-a sequence by diagnostic

restriction analysis and automated sequencing, the plasmidknown antiproliferative effect against cancer cells in vitro,25including bladder tumour cells,26 its ability to induce LAK constructs were introduced into M. smegmatis by electropor-

ation (easyjecT PLUS; Flowgen Instruments Ltd, Lichfield,activity27 and to regulate MHC class II and ICAM-1expression.19,28,29 Mycobacterium smegmatis was selected as it Staffs.,UK ). Aliquots of M. smegmatis [200 ml at #5×108

clony-forming units (CFU )/ml ], washed and resuspended inis less pathogenic than BCG and is more easily handled in thelaboratory, having a doubling time of 2–6 hr as opposed to 10% glycerol (Sigma Chemical Co., Poole, Dorset,UK ), were

transferred to precooled electroporation cuvettes (Flowgen)22 hr for BCG. In addition, we have shown it to be almost200 times more potent with respect to inhibiting bladder and 1–2 mg of purified vector construct DNA was added.

Transfection reactions using empty, circular pMOD vectorstumour cell growth in vitro (unpublished observations).Here, we present details of gene transfection into myco- were conducted to produce direct controls for subsequent

assays. Electroporation conditions were set at 2·5 kV, 25 mFbacteria using a shuttle plasmid strategy, analysis of theconcentration and biological activity of the human recombi- and 700 V, this gave a time constant of #17 milliseconds. To

the electroporated mycobacteria, 1 ml of Middlebrook 7H9nant protein and results of in vitro investigations into tumourICAM-1, human leucocyte antigen (HLA)-DR and cytokine liquid medium containing 10% Middlebrook albumin–dextrose

complex (ADC ) enrichment (Difco Laboratories, Detroit,induction and killing by wild-type and recombinant M. smeg-matis clones. MI ) was added and the cell suspension was incubated for 2 hr

before plating onto Middlebrook 7H11 agar (Difco) containing0·5% glycerol, 10% Middlebrook ADC enrichment and

MATERIALS AND METHODS30 mg/ml kanamycin. Following 3 days incubation at 37°,transfectants were selected by their antibiotic resistance andGene amplification

Primers for amplification of the mature peptide sequence of their fluorescence under ultraviolet illumination (pMOD12series).human TNF-a were designed incorporating appropriate

restriction enzyme sites for unidirectional cloning into themultiple cloning site of Escherichia coli–mycobacteria shuttle Analysis of recombinant protein

A number of kanamycin-resistant M. smegmatis colonies wereplasmids pMOD8 and pMOD12gfp. The TNF-a cDNA wasamplified using reverse transcription–polymerase chain reac- used to establish liquid cultures and growth was monitored by

absorbance at 600 nm. Supernatant samples from mid-logtion (RT-PCR) on mRNA from human donor lymphocytesstimulated with 4b-phorbol 12b-myristate 13a-acetate (PMA). phase cultures were analysed using sodium dodecyl sulphate–

polyacrylamide gel electrophoresis (SDS–PAGE) and WesternIsolation of mRNA was achieved on oligo-dT spin columns(Qiagen Ltd, Crawley, West Sussex, UK). blotting techniques, probing with monoclonal anti-[HA]

(Boeringer Mannheim, Sussex, UK) and anti-human(hu)TNF-a antibodies (R & D Systems Europe Ltd,Vector technology

The pMOD8 and pMOD12gfp shuttle expression vectors used Abingdon, Oxon, UK) and enzyme-linked immunosorbentassay (ELISA; Quantikine HS immunoassay kit; R & Din this study were a gift from Dr M. O’Donnell, Division of

Urology, Beth Israel Hospital, Boston, MA. These include Systems) for the presence of huTNF-a. An L929 bioassayemploying recombinant huTNF-a standards (R & D Systems)separate origins of replication for both E. coli and myco-

bacteria, an a-antigen signal peptide sequence driven by the was subsequently undertaken to assess the biological activityof the recombinant protein.31heat-shock protein (hsp 60) mycobacterial promoter and the

12CA5 epitope derived from the influenza haemagglutinin(HA) protein which serves as a tag for the detection of the Mycobacterial culture

Two transfected M. smegmatis clones, referred torecombinant protein. The pMOD series of vectors also carrya kanamycin resistance gene for selection of transformants. In as pMOD8–TNF-a and pMOD12gfp–TNF-a, demonstrated

to be the most efficient secretors of recombinant humanaddition, the pMOD12gfp vector incorporates a second pro-moter (hsp 70), two multiple cloning sites and a gene encoding TNF-a, were chosen and grown in 500 ml Middlebrook 7H9

medium containing 0·2% glycerol, 0·05% Tween-80 (Sigma)the jellyfish (Aequorea victoria) green fluorescent protein

© 1999 Blackwell Science Ltd, Immunology, 96, 114–121

J. L. Haley et al.116

and 30 mg/ml kanamycin supplemented with ADC enrichment. a window and 5000 events were accumulated using logarithmicamplification of fluorescence intensity.Empty vector (mock-transfectants) and wild-type control

M. smegmatis cultures were established concurrently. Wild-typeProliferation assaycultures were grown in the absence of antibiotics. Large-scaleCell proliferation was monitored by measuring tritiated thymi-cultures were incubated at 37° for 2–5 days and the myco-dine incorporation into the DNA of replicating cells. The fourbacterial cells were then washed to remove the 7H9 mediumbladder tumour cell lines were seeded at 1000–5000 cells/welland antibiotics in RPMI-1640 medium supplemented within 96-well flat-bottomed plates (Costar UK Ltd, High25 m HEPES, 2 m -glutamine, 1 m sodium pyruvate andWycombe, Bucks., UK). Optimum concentrations of wild-5% heat-inactivated fetal calf serum (FCS) (all from Sigma).type or transfected mycobacteria (5×104 CFU/ml ) were addedDilutions of the resuspended cultures were prepared for CFUto mid-log phase cultures. At 16 hr prior to the harvest time-determination, 100 ml of each dilution was plated onto 7H11point, 0·5 mCi methyl [3H]thymidine (Amersham Internationalagar and the resultant colonies were counted after 3 daysplc, Little Chalfont, Bucks., UK) was added to each wellincubation at 37°. The number of CFU/ml was calculated inunder sterile conditions. The cells were trypsinized and reco-each case. The remaining M. smegmatis cells were frozen invered at 24, 48 and 72 hr with a semiautomatic cell harvesteraliquots at −70°.(Skatron, Lier, Norway). Filter discs were dried and countedusing a scintillation analyser (Tri-Carb 1900CA; CanberraCell culturePackard, Pangbourne, Berks., UK).Four human transitional cell carcinoma lines, EJ18, MGH-U1,

RT4 and RT112, a gift from Dr J. Masters, Institute ofCytotoxicity assayUrology, London, were employed as an in vitro model forLysis of bladder tumour cells by M. smegmatis-activated killerbladder cancer. The cell lines were maintained in completecells (designated as SMAK cells) was investigated in a chro-RPMI-1640 medium without antibiotics at 37° in a humidifiedmium (51Cr) – release assay. Effector cells were induced byatmosphere of 5% CO2 in air. Adherent monolayers werestimulating PBMC, isolated from normal donor buffy coat byroutinely recovered by trypsinization with 0·5 mg/ml trypsin,density gradient centrifugation on Lymphoprep (Nycomed,0·2 mg/ml tetrasodium ethylenediamine tetraacetic acidOslo, Norway), with 2×102 CFU/ml wild-type or recombinant(EDTA; Sigma) and manipulations were carried out in a classmycobacteria over a period of 4–6 days. LAK cells wereII microbiological safety cabinet (Envair Ltd, Rossendale,generated concurrently by incubating PBMC from the sameLancs, UK).donor with 1000 U/ml recombinant IL-2 (PeproTech EC Ltd,London, UK). Target cells were labelled by adding Na251CrO4Measurement of cytokine induction(Amersham) at 100 mCi per 106 tumour cells in completeThe effects of wild-type and recombinant M. smegmatis onRPMI-1640 medium and incubating under normal cell culturethe expression of cytokines by cultures of bladder tumour cellsconditions for 1 hr. The cells were then washed three times inand peripheral blood mononuclear cells (PBMC ) were investi-complete medium, resuspended at a concentration of 5×104gated. Supernatant samples from naive cell populations andcells/ml and seeded into 96-well V-bottomed microtitre platescultures incubated with M. smegmatis clones were collected at(Costar) at 100 ml per well. Effector cells were added to achieve72 hr (tumour cells) or at 6 days (PBMC ) and analysed fora previously determined optimum effector5target ratio of 4051TNF-a concentration and the presence of other cytokinesproducing a final volume of 200 ml. After centrifugation at(IL-1b, IL-2, IL-6, IL-8, GM-CSF and RANTES) using100 g for 30 seconds, the plates were incubated for 4 hr,commercially available ELISA assays (R & D Systems).centrifuged again at 100 g for 5 min and finally 100 ml ofsupernatant from each well was transferred into tubes forCytofluorometric analysismeasurement in a gamma-counter.Single-colour immunofluorescence was conducted on bladder

Results are calculated as percentage specific toxicity usingtumour cells stained with monoclonal antibodies to humanthe formula:ICAM-1 (BBIG-I1; R & D Systems) or HLA-DR (fluorescein

isothiocyanate-conjugated CR3/43; DAKO Ltd, Ely, (experimental release−spontaneous)

(total release−spontaneous release)×100Cambridge, UK). The cells, incubated with an initial concen-

tration of 5×104 CFU/ml mycobacteria, recombinant TNF-aSpontaneous release was measured by determining the counts(positive control ), or with medium alone, were harvested atin supernatants from target cells cultured in medium alone24, 48 and 72 hr, washed in phosphate-buffered saline, pH 7·4,and total release was measured by determining counts fromsupplemented with 1% heat-inactivated FCS and 0·01% sodiumtarget cells lysed with 1% Triton X-100 (Sigma).azide (all from Sigma) and incubated with optimal concen-

trations of primary antibody for 30 min at 4°. The binding ofthe unconjugated ICAM-1 antibody was detected by incubat- RESULTSing the washed cells with goat anti-mouse IgG (F(ab∞)2- Expression and secretion of biologically active recombinantfluorescein isothiocyanate-conjugated; Sigma) at 4° for a

TNF-afurther 30 min and controls were incubated with secondaryantibody alone. Following antibody incubations, the cells were The secretion of TNF-a was initially verified by probing

nitrocellulose blots of SDS–PAGE-separated culture super-washed and resuspended in washing buffer containing 1%formaldehyde (Fisher Scientific UK, Loughborough, Leics., natants with antibodies to the 12CA5 epitope sequence tag

and to human TNF-a. These studies revealed the presence ofUK). Fluorescence was measured on an EPICS-XL flowcytometer (Coulter, Luton, UK). Live cells were gated within bands at 17 000 MW in transfected culture supernatants

© 1999 Blackwell Science Ltd, Immunology, 96, 114–121

Enhancing mycobacterial immunotherapeutic potential by genetic manipulation 117

Table 1. Cytokine expression by tumour cell cultures incubated for 72 hr with medium alone or with 5×104 CFU/ml wild-type or recombinantM. smegmatis clones

TNF-a IL-1b IL-6 IL-8 GM-CSF RANTES TGF-b1

EJ18Medium alone × × 188 144 × × 501Wild-type cells × × 2774 3860 × × 147TNF-a-transfected 23 900 × 6223 7850 30 × 60Vector-transfected 40 × 4250 6470 × × 65

MGH-U1Medium alone × × × 280 × × 830Wild-type cells × × × 3000 × × 114TNF-a-transfected 9700 × 1251 9090 54 × 120Vector-transfected × × × 6120 39 × 143

RT4Medium alone × × × 59 × × 158Wild-type cells × × 87 2670 × × 120TNF-a-transfected 9400 37 394 33 510 306 × 107Vector-transfected × × 134 2680 × × 77

RT112Medium alone × × × 825 40 × 139Wild-type cells × × × 10 190 831 × 173TNF-a-transfected 9100 × × 21 140 3215 × 72Vector-transfected 58 × × 17 270 1570 × 172

Results show the TNF-a-secreting mycobacteria is a more effective inducer/up-regulator of IL-6, IL-8 and GM-CSF. Average values fromduplicate determinations are presented and × denotes a concentration below the sensitivity of the ELISA assay (TNF-a <4·4 pg/ml;IL-1b<1 pg/ml; IL-6<0·7 pg/ml; IL-8<10 pg/ml; GM-CSF<2·8 pg/ml; RANTES <5 pg/ml ). A typical result is presented from a minimumof two separate experiments. With all cell lines in all experiments the highest TNF-a, IL-6, IL-8 and GM-CSF levels were always observed inthe supernatants from tumour cells treated with TNF-a-transfected mycobacteria.

corresponding to the human recombinant protein. ELISA natants from 3-day tumour cell cultures incubated alone orwith 5×104 CFU/ml wild-type, vector-transfected or TNF-a-analysis confirmed levels of TNF-a in the same culture super-

natant samples ranging from 13 to 80 ng/ml following 7 days transfected M. smegmatis.TNF-a concentrations in supernatants from MGH-U1,of culture. No TNF-a was detected by either technique in

supernatants from bacterial cultures transfected with empty RT4 and RT112 incubated with TNF-a-transfected M. smeg-matis reflected levels obtained from 5×104 CFU/ml of thevector. Higher secretion levels were found to be produced by

clones containing the single promoter/cloning site (pMOD-8) TNF-a-transfected clone grown alone without antibiotic selec-tion. This suggests that the TNF-a was secreted exclusivelyvector construct. Supernatant samples from the most efficient

TNF-a-secreting M. smegmatis clone (pMOD8–TNF-a), from the recombinant mycobacteria. For EJ18 however, aconcentration almost 2·5 times higher indicates an inductionseeded at 5×104 CFU/ml in RPMI medium without anti-

biotics, were collected at 24, 48 and 72 hr and found to contain of TNF-a protein from the tumour cells themselves. Lowconcentrations of TNF-a were also found in supernatantsbiologically active human TNF-a which increased in concen-

tration from 230±65 pg/ml (1·6±0·5 IU/ml ) to 9·8± from EJ18 and RT112 cell lines incubated with M. smegmatistransfected with empty vector. As no TNF-a was detected0·7 ng/ml (69±14 IU/ml ) over 3 days.from mock-transfected M. smegmatis cultured in isolation orfrom tumour cells cultured alone or with wild-type clones, it

The effects of wild-type and recombinant M. smegmatis onappears TNF-a is released by the tumour cells in response to

tumour cells in vitrothe presence of the vector.

No significant induction of IL-1b (except RT4 cells incu-In previous studies we have shown that BCG directly effectshuman bladder tumour cell lines in vitro, inducing and/or bated with TNF-a-transfectant) or RANTES was seen, how-

ever, a distinct pattern emerged for IL-6, IL-8 and GM-CSFup-regulating ICAM-1 expression,17 the secretion of cyto-kines13–15 and inhibiting tumour cell growth.32 We have there- expression. We were able to show greater induction/up-

regulation of expression from cell lines infected with recombi-fore extended these investigations to study the effects of wild-type and recombinant M. smegmatis on these parameters. nant M. smegmatis strains compared with wild-type and levels

were always higher in supernatants from cells subjected to theTNF-a-transfectants. A reversal of this trend was demon-

Cytokine induction by wild-type and transfected mycobacteriastrated for TGF-b1, with three out of four cell lines infectedwith all mycobacteria preparations producing lower levels ofTable 1 presents the concentrations of TNF-a, IL-1b, IL-6,

IL-8, GM-CSF, RANTES and TGF-b1 detected in super- TGF-b1 compared to tumour cells cultured in medium alone.

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J. L. Haley et al.118

% cells expressing ICAM-1

Medium

TNF-a transfected

Medium+TNF Ab

TNF-a transfected+TNF Ab

Mock transfected

Mock transfected+TNF Ab

151050 2520 30

Figure 2. The quenching of ICAM-1 expression by anti-huTNF-aantibody. MGH-U1 tumour cells were incubated for 48 hr withmedium, TNF-a-transfected or mock-transfected M. smegmatis clonesboth with and without the addition of 5 mmg/ml anti-huTNF-a. Themean percentage ICAM+ cells±standard deviation are calculatedfrom triplicate determinations.

Anti-proliferative effects of wild-type and transfectedmycobacteriaTime (h)

24 48 72

% c

ells

exp

ress

ing

ICA

M-1

80

40

20

0

EJ18

24 48 72

24 48 72 24 48 72

MGH-U1

RT4 RT112

80

60

20

0

25

20

5

0

80

60

20

0

40

1540

Medium alone

Wild-type

TNF-a transfectant

Vector transfectant

10

60

Figure 1. The effect of wild-type and recombinant forms of M. smeg- Our early observations revealed the antiproliferative potencymatis on ICAM-1 expression. Bladder tumour cells were stained of M. smegmatis to be up to 200 times that of BCG. Usingfollowing 24, 48 and 72 hr incubation with 5×104 CFU/ml myco- initial concentrations of 5×104 mycobacterial CFU/ml, abacteria and ICAM-1 levels were compared with cells incubated with gradual reduction in tumour cell proliferation could be demon-medium alone. Results show significantly greater up-regulation of strated over 72 hr. This concentration was employed in allICAM-1 expression by the TNF-a-transfectant compared with wild- subsequent studies.type and vector-transfectant. A typical experimental outcome is pre-

Results for all four transitional cell carrcinoma cell linessented for each cell line from four similar repeat determinations andshow a similar trend. Both TNF-a-transfected and mock-means±standard deviations are calculated from triplicate assessments.transfected M. smegmatis were more effective than wild-typemycobacteria in inhibiting tumour cell replication. The TNF-atransfectant was slightly, but consistently, more effective thanthe mock-transfectant in this regard (Fig. 3).It should be stressed that similar effects were reproducibly

observed on a minimum of two to five separate occasionsdepending on the cytokine. The effects of SMAK cells

Due to its remarkable potency and rapid replication rate,which resulted in overgrowth of cultures accompanied by poor

Induction of ICAM-1 and HLA-DR expressionsurvival of lymphocytes, initial concentrations of2×102 CFU/ml M. smegmatis were chosen to study thePrevious studies from our laboratory have reported the consti-

tutive surface expression of ICAM-1 by the four bladder activation of PBMC over 4–6 days. Resultant SMAK effectorcells produced specific cytotoxicity values of 21–43% (day 5)tumour cell lines to be in the range 4–80% positive cells,19

with the greater levels in higher grade tumours dependent in the four bladder cell lines, compared with 55–87% for LAKcells. However, no significant enhancement of cytotoxicty wasupon cell density and autocrine factors.33 The current study

revealed up-regulation of ICAM-1 expression on all four cell observed with recombinant mycobacteria compared with wild-type (Table 2).lines following infection with the TNF-a-transfected M. smeg-

matis (Fig. 1). Wild-type and vector-transfected mycobacteria No cell surface phenotypic differences between wild-typeand recombinant SMAK cells were revealed by flow cytometrywere shown to be less effective at augmenting cell surface

ICAM-1 levels in every case and failed to induce any expression using monoclonal antibodies to CD3, CD4, CD8, CD25,HLA-DR and cd. In fact, the number of CD25+ and HLA-on the RT4 cell line. Additional experiments on the MGH-U1

cell line indicated that the induction of ICAM-1 by TNF-a- DR+ cells remained unchanged from non-activated PBMCpopulations. All M. smegmatis-activated killer cells however,transfected M. smegmatis could be largely, though not com-

pletely, inhibited by inclusion in the culture of excess neutraliz- demonstrated an increase in CD8+ cells.Cytokine measurement showed notable induction of IL-6,ing antibody to TNF-a (Fig. 2).

Incubation of the RT4 cell line with TNF-a-secreting M. IL-8 and RANTES but no significant difference was observedbetween the WT-SMAK and recombinant-SMAK cells. IL-2smegmatis also produced an increase in MHC class II

expression (2·6-fold) which was not achieved with either wild- production was not detected from any of the SMAK cellcultures.type or mock-transfected species (data not shown).

© 1999 Blackwell Science Ltd, Immunology, 96, 114–121

Enhancing mycobacterial immunotherapeutic potential by genetic manipulation 119

no N-glycosylation sites, its protein sequence gives rise to asingle disulphide bond34 and it appears non-toxic to themycobacterial host. ELISA and bioassay analysis of super-natants from TNF-a-transfected M. smegmatis cultures haveconfirmed high levels of bioactive recombinant proteinsecretion.

An in vitro model based on four human bladder transitionalcell carcinoma cell lines was chosen to examine differences inimmunomodulatory activity between wild-type and recombi-nant M. smegmatis clones. We have demonstrated significantdifferences in tumour response to wild-type and transfectedstrains. TNF-a-secreting mycobacteria proved to be reproduci-bly more effective in augmenting/inducing proinflammatorycytokine secretion (IL-1b, IL-6, IL-8, GM-CSF) and cellsurface ICAM-1 expression. We have also shown enhancementof mycobacterial antiproliferative properties as a result ofTNF-a transfection but failed to observe any differences inability to induce SMAK activity. Interestingly, our experimentsalso reveal that mock-transfected mycobacteria possess greaterimmunostimulatory properties than the wild-type.

These studies with TNF-a-transfected M smegmatis extendprevious observations on the enhanced immunomodulatoryactivities of mycobacteria transfected with other human androdent cytokine genes.22–24

Time (h)

24 48 72

Inco

rpor

atio

n of

triti

ated

thym

idin

e (c

.p.m

.×10

4 )

30

20

10

0

EJ18

24 48 72

24 48 72 24 48 72

MGH-U1

RT4 RT112

8

6

2

0

20

15

5

0

4

3

1

0

4

102

Medium alone

Wild-type

TNF-a transfectant

Vector transfectant

Figure 3. A comparison of wild-type and recombinant mycobacterial Experiments demonstrating increased up-regulation ofantiproliferative potency measured by incorporation of [3H]thymidine cytokine expression by TNF-a-transfected mycobacteriaby bladder tumour cell lines. Cells were incubated with medium alone appear to confirm documented views concerning the role ofor with 5×104 CFU/ml wild-type, TNF-a-transfected or vector- TNF-a in immune modulation. Most of the cytokines investi-transfected M. smegmatis over 72 hr. Results show TNF-a-transfected gated here (IL-1, IL-6, IL-8, GM-CSF) are known to beM. smegmatis clones are significantly more effective than wild-type induced by TNF-a or at least stimulated by the same exogenousand marginally more effective than vector-transfected strains at arrest-

or endogenous signals.35 It is not unreasonable to assume thating tumour cell growth. A typical result is presented for each cell linethe enhanced ability of TNF-a-transfected M. smegmatis toshowing the mean±standard deviation of six replicate wells. Eachinduce cytokines such as IL-8 (which enhances chemotaxisexperiment was performed on four separate occasions with reproduc-and activation of neutrophils) and GM-CSF (which increasesible results.production of granulocytes and macrophages) should endowit with superior anti-tumour properties in vivo. Of additionalTable 2. The induction of activated killer cells (SMAK cells) by wild-potential benefit in this regard are the observations thattype and transfected M. smegmatisM. smegmatis (transfected or otherwise) down-regulates the

Target cells production of TGF-b1 by tumours. This cytokine is over-produced in many tumours and may provide growth advan-

Effector cells EJ18 MGH-U1 RT4 RT112 tage due to its stimulation of extracellular matrix formationand its local immunosuppressive effect on cytotoxic T cells

LAK 87 62 76 71 57 66 55 64and natural killer cells.36,37

WT-SMAK 35 26 32 34 24 28 30 33It has been previously reported that cytokines found in theTNF-a-SMAK 37 34 36 39 30 37 31 40

urine of patients receiving BCG immunotherapy augmentvector-SMAK 39 21 41 37 32 29 33 43ICAM-1 and HLA-DR expression on bladder tumour cells.26Our results show wild-type M. smegmatis to be an extremelySpecific toxicity values (%) are presented for effector cells activatedeffective up-regulator of ICAM-1, verifying the suitability ofby wild-type M. smegmatis (WT-SMAK), TNF-a-transfected M.

smegmatis (TNF-a-SMAK) and mock-transfected M. smegmatis this strain as a model for BCG. In accord with previous(vector-SMAK) for 5 days. The cytotoxic activity of simultaneously observations, augmentation of ICAM-1 by the TNF-a-generated IL-2-induced LAK cells is included for comparison. No secreting strain is likely to promote increased recognition ofsignificant difference in cytotoxic properties was observed between the tumour cells by activated leucocytes via ICAM-1–LFA-1wild-type and recombinant M. smegmatis-activated killer cells. Two interactions.18–20separate experimental results are presented and values represent the

An increase in MHC class II expression on RT4 cells inaverage of four replicate wells.response to TNF-a-transfected M. smegmatis suggestssecretion of the recombinant protein may engage a second

DISCUSSIONpathway involving presentation of mycobacteria-derived anti-gens by the MHC-expressing tumour cells producing a specificMycobacterial strains incorporating the genetic sequence of

human TNF-a have been engineered using an extra- T-cell response.21The antiproliferative activities of mycobacteria themselveschromosomal plasmid system. TNF-a conforms to all predict-

able criteria for efficient mycobacterial expression.24 It contains and of a number of immune system-derived cytokines,

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J. L. Haley et al.120

including TNF-a, have been described in cultured bladder organisms (2×102 CFU/ml ) used in these studies remains tobe established.tumour cells.26,32 Wild-type M. smegmatis exhibited remarkably

potent inhibitory effects on tumour cell growth. Early experi- Our results illustrate the immunomodulatory potential ofgenetically modified mycobacteria. Production and secretionments demonstrated the effect of 5×104 CFU/ml M. smegmatis

to be equivalent to #1×107 CFU/ml wild-type BCG of cytokines at the primary tumour site, thereby mimickingphysiological expression, has advantages over systemic admin-and only modest improvements in cytostatic ability could be

attributed to TNF-a secretion. istration in that comparatively small concentrations of theseextremely toxic molecules are introduced locally into theIn all of the experiments described, there is evidence of an

improvement in immunological activity of mycobacteria patient. We have presented evidence of enhancement of the invitro immune response and tumour response to mycobacteriatransfected with vector alone. The reason for this is unclear

but modification of growth characteristics as a consequence secreting TNF-a. This may be of significant clinical benefit inthe improvement of mycobacterial immunotherapy protocolsof the transfection protocol has been ruled out as no differences

in replication rate between wild-type and recombinant species and animal models are currently being developed to determinein vivo therapeutic prospects.were apparent in the absence of antibiotic selection. However,

it is not unexpected that the vector-transfectant generatesgreater adhesion molecule expression and antiproliferative ACKNOWLEDGMENTSactivity given the higher levels of cytokines induced from

The authors would like to thank Dr M.A. O’Donnell for the generoustumour cells cultured with mock-transfected mycobacteriadonation of expression vectors, Mr J. Black for conducting the TNF-acompared with wild-type. At the present time we can onlybioassays and Drs T. Paterson and J. Innes for assistance withspeculate on why mock-transfected M. smegmatis is moreelectroporation protocols. J. L. Haley, D. G. Young, A. Alexandroffactive than wild-type with respect to a number of the effectsand K. James gratefully acknowledge the generous financial supportwe have investigated. One remote possibility is that the use ofof the Scottish Hospitals Endowment Research Trust (SHERT).

vectors containing hsp promoters may result in the induction A. M. Jackson wishes to acknowledge the support of the Imperialor enhanced production of hsp by the tumour cells themselves Cancer Research Fund (ICRF ) and the Association of Internationalwhich in turn leads to enhanced immunomodulatory and Cancer Research (AICR).cytotoxic effects, though this remains to be tested. Neverthless,these experiments highlight the importance of including such

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