IFN-γ production by Mycobacterium tuberculosis stimulated human CD30+ αβ and γδ T cells

IFN-γ production by Mycobacterium tuberculosis stimulated human CD30+ αβ and γδ T cells

Mycobacterial infections 25 June 1997 - Poster presentations performing the same in vitro experiments wtth RAG-2-knockout mouse spleen cells. These ...

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Mycobacterial infections

25 June 1997 - Poster presentations

performing the same in vitro experiments wtth RAG-2-knockout mouse spleen cells. These results showed that the administration of anti-asial&Ml serum depletes cytotoxic cells but not the IFNy secreting Thyl.2+CD3- cells that confer protection against M. avium infections. NK cells seem to be heterogeneous, with an asialo GMl+ population that is cytotoxic and an anti-asialo GM1 insensitive population that secretes IFNy.

P.4.11 .12

Picolinic acid, a catabollte of L-tryptophan, induces intra-macrophaglc bacterlostasls In hfycobacterium avium

Teresa Pais, Rui Appelberg. Centm de Citologia Experimental, Universily of Porto, Portugal One of the mechanisms of antimicrobidal and tumodcidal actfvity of macrophages implies the induction of inddeamine 2,3dioxigenase (IDO) by IFN-]I. This enzyme is responsible for the degradation of L-tryptophan during the macrophage activation. Plcdinic acid (PA) is a terminal metaboiiie of L-ttyptophan that has been detected in body fluids and identified as a zinc binding ligand present in human milk. It has been shown that PA is a cestimulatoty agent for the induction of tumottcidal activity and synergises with IFN-y for the induction of nittic oxide syntethase and TNFa mRNA expression in the macrophage cell line ANA-1 and peritoneal macrophages. It also forms extremely stable complexes with iron and affects imn metabolism in NRK cells. We demonstrate that PA has a bacterfostatic effect in the growth of Mycobac+etium avium in axenic medium, the same effect is not seen with the non chelator isomer nicottnic acid or with quinolinic acid. When added to bone marrow-derived macrophages, PA inhibits the growth of M. atium strain 25291 ST at the same level as IFN-y. The addition of PA together with IFN-y induces complete bactetfostasis showing that PA is also a co-stimulatory agent for the macrophage anti-mycobacterfal activity. This antimicrobial activity of PA may be due to an effect in the iron metabolism inside the macrophage as well as to other regulatory effects.

1P.4.11 .13

Neutrophil chemlluminescence (CL) In tuberculosis

’ (TB) contacts and TB patlents K. Zabuska, A. Ponahajba, H. Niemirowska, E. Skopifiska-R6iewska. The Institute of Tuberculosisand Lung Diseases, Warsaw, Poland Introduction: The phagceytosis process is an important defense mechanism in TB. The creation of reactive oxygen species (ROS) in activated neutrophils during “respiratory burst” is accompanied by photon emission which can be measured by CL test. The aim of our work was to assess the phagocytic activity of TB amtacts and TB patients neutrophils by means of CL test. Material and Methods: The luminol enhanced CL of neutrophils isolated from peripheral blood on Gradisd G gradient was assayed using LKB 1251 Luminorneter in the following three groups of people aged 20 to 45 yrs: i) 16 persons infected with M. tuberculosis eB cot&&), ii)-16 untreated patients (pts) with newly diagnosed active pulmonary TB. iii) 22 healthy blood donors. The TB conta&s g&up consisted of nurses from TB wards, with excessive Mantoux reaction (> 15 mm in diameter) and nonal chest X-ray. Resuh The neutrophils CL of TB contacts was significantly decreased as compared to TB pts and healthy donors (p < 0.001). The average values of Cl,, and CLlsmin (integrated photon emission measured during 15 min) of FMLP stimulated neutrophils in those groups were respectively in mV’s: i) 19.8 f 2.7, 5100 f 550; ii) 49.1 f 6.6, 17800 f 2900; iii) 44.5 f 4.2, 14100 f 1400. Also the studies of CL activity of FMLP stimulated granulocytes in whole venous blood using B-liquid scintillation counter in 30 TB contacts showed the impairment of CL in TB contacts as compared to 30 healthy donors (14600 f 1740 cmp/l@ neutrophils vs 50600 f 2260 cpm/ld, p c 0.001). Furthermore the neutrophil CL actfvity after specific stimulation by opsonised Bacilli Calmette-G&in was studied in 5 TB pts during pilot studies. The CL activity was varied and equalled to 20-3396 (3 pts) and 172% and 264% (2 pts) of the value of CL activity in response to FMLP stimulation. Due to different extent and stage of changes in those 5 TB pts further studies encompassing larger number of pts are necessary. Conclusions: - The impainent of granukxzyte CL in whole blood of TB contacts as well as their isolated neutrophils suggests the higher tisk of progression from M. tuberculosis infection into fully blown disease. -The neutrophil CL assay may aid the standard procedures in screening for groups with an increased risk of developing the disease. - Varied CL activity after specific stimulation of neutrophils in TB patients requires further studies.

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1P.4.11 .14 1 Tuberculosis, mycolic acids and lnterleukin 12 expression in mice A. Lenaetts, A. Pretorfus, J.A. Verschoor. Department of Biochemistw University of Pretoria, Pretoria 0002, South Africa with the development Infection with Mycobacfedum tubemu/osis is bated of protective immunity, which is ptimarily mediated by CD4 T cells and mononuclear phagocytes. An important role in cell mediated immunity is played by 11-12, an earty proinflammatory cyioklne produced by macmphages. In murine models administration of II-12 induces protection against intracellular micro-organisms such as M. tubercoosis. Among other mycobactetfal components immunostimulatory properties have been ascribed to a cell wall component, mycolic acids (MA). MA are long chain fatty acids and compose about 40% of the dry weight of M. tubercu/osis. MA are found to have a unique property by stimulating CD4(-) CD8(-) T cells upon CD1 presentation. The imunogenic activity of MA, besides double negative T cell stimulation, is the aim of our investigation which focuses on cytokine expression and more specifically the expression of IL-12. The expression of the inducible p40 subunit of IL-12 in two inbred mouse strains (the susceptible Balblc strain and more resistant C57Bl6 strain) was compared in diierent organs upon intravenous TB infection. Additionally, in a preliminary experiment IL12 p40 expression was studied after 4 h, 8 h, 17 h and 41 h in both mouse strains after injections with purified MA. IL-12 mRNA expression was measured in comparison to the B-actine household gene by semiquantttatfve RT-PCR and subsequently quantified by competitive RT-PCR using a competitor plasmid (gift K. Best. University of Tulane, USA). PCR results showed a higher IL-12 expression in C57Bl6 mice compared to the Balb/c strain in all investigated organs. IL-12 expression in these mouse strains correlate with resistance to M. tubercu/osis infection. The preliminary MA experiments will be confirmed and presented.

P.4.11 .15

Antlbodles against mycollc acids purlfled from Mycobacterium tubenx~losis

M.A. Goodrum, D.G.R. Siko, H. Macmillan, J.A. Verschoor. Deparfment of Biochemistw, University of Pretoria, Pretoria 0002, South Africa Tuberculosis is reaching epidemic proportions, mainly due to the occurrence of dNg resistance and AIDS. Diagnosis, treatment and/or therapy requires knowledge on the role that each chemical component of the pathogen plays in disease. Mycollc acids, the major lipids of the cell wall of mycobacteda, appears to influence disease by stimulating CD4/CD8 double negative T cells’. These high molecular weight, waxy molecules characteristic of Mycobacteria occur as mixtures of different types, esterifled to mycobacterial cell wall carbohydrates. In this paper, the immunogenicity of purified mycolic acids towards stimulation of antibody secretion was investigated. Mycolic acids, extracted by saponification in potassium hydroxide methanol solution, were purified by the Counter Current Distribution (CCD) technique and tested for biologically activity In respect of their ability to stimulate double negative human T cells. Pure mycolic acids were adsorbed on bovine serum albumin and immunised into Balb/c mice. Attempts were made at producing monoclonal antibodies from the immune mouse spleens. Human TB patient sera were screened for the presence of mycolic acids specific antibodies. Mouse antisera obtained 83 and 202 days into the immunisation program were tested in ELISA using gelatin-mycolic acids conjugate as solid phase antigen. Stronger responses were shown on mycolic acids-gelatin than on gelatin coated plates and exhibited maturation towards the IgG1 and lgG2 isotypes and increased signal strength. The antibodies remained of low affinity, however, which prevented their detection in monoclonal antibody supematants. Antibodies against mycolic acids could not be detected among a limited number of human TB patient antisera. [I] Beckman, EM eta/. (lQQ4) Recognition of a lipid antigen by CDI-restricted &J+T cells. Nature 372: 691494.

1P.4.11.16 1 1FN-y production by Mycobacfer/um fubercu/os~s stimulated human CD30* ap and 76 T cells MartinE. Munk, Stefan H.E. Kaufmann. Max-P/an&-/nstifute for /nkction Bidogy; Monbijoustr 2, 10117 Berlin, Germany Introduction: CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and evidence has been presented that activated CD4+CD45Ro+ T cells of T-helper (Th) 2 type selectively express CD30. Mycobacterium tuberculosis, a facuitative Intracellular bacterium capable of replicating in resting macrophages, is a potent inducer of interferon-y secretion by Thl cells. Therefore, we investigated the expression of CD30 on mycobacterfal stimulated T cells from healthy donors and tuberculosis pattents. Results: 1) increased CD30 expression by M. tubercukzsiestimulated a/b and y6 T cells and elevated numbers of CD30+ a/p T cells in tuberculosis pleuritis were detected by cytofluorfmetry; 2) tuberculosis patients showed in-

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25 June 1997 - Poster presentations

Mycobacterial infections

tense accumulation of CD30+ cells in the vicinity of the bronchus and along the surrounding inflammatory tissue in immuno-histological sections; 4) freshly isolated PBMC from tuberculosis patients collected at different time points after initiation of anti-tuberculosis chemotherapy did not encompass significant levels of CD30+ cells dutfng clinical improvement; 5) surface CD30 expression was associated with intracytoplasmic IFN-y expression and IFN-y production by M. f&ercu/o&stimulated a//3 and yS T cells. Conclueions:Although M. tuberculosis is a typical stimulator of Thl cells it is also a potent inducer of CD30 expression. Thus, our results argue against exclusive correlation of CD30 expression with Th2 cell responses.

P.4.11.17

Induction of cellular immunity against Mycobacferium tuberculosis

D.P.A.J. da Fonseca, A.F.M. Verheul, H. Snippe. Eijkman-Winkler institute for Microbiology Infectious Diseases and Inflammation, Utrecht University; Utrecht, The Netherlands

Introduction:BCG (Bacillus Calmette-Guerin) is the currently used vaccine against tuberculosis. It mainly activates CD4+ T-cells but fails to activate CD8+ T-cells. A good induction of cellular immunity, covering both CD4+ and CD8+ T-cell activation, is a requirement for providing protection against infection with A&wbacfetium tubercukxis. This study focuses on the development of methods for inducing optimal cytotoxic T-cell (CTL) responses against peptide epitopes. Cytolytic T-cell epitopes were identiied by the following approach: 1) peptides (7-10 aa) designed from the 19 kD and 38 kD mycobacterfal proteins sequence, with known class I binding motifs, were synthesized; 2) their ability to up regulate class I MHC expression on RMA-S (H-2b) cells was investigated; 3) lipid tails were attached to the identified peptides and their ability to induce CTL in combination with adjuvants was studied in viw. Mate.rlal and Methods:RMA-S (H-2b) cells were incubated with 19 kD and 38 kD peptides and lipopeptides with known class I MHC mouse binding motifs (H-2Ld and H-2Db for the 19 kD and H-Db for the 38 kD), stained for class I MHC expression using a specific mAb and analyzed by flow cytometry. Mice were immunized with the peptides or lipopeptides in combination with Incomplete Freund’s Adjuvant (IFA). Spleens were isolated at day 7-14, restimulated in vitro for 8-8 days and a standard 51Cr release assay was performed. Results: Five different peptides and their corresponding lipopeptides were able to up regulate class I MHC molecules (3 from 19 kD, 2 from 38 kD). Experiments perfoned so far, demonstrated that one of the identified peptidss of the 38 kD protein is a CTLepitope. Moreover, the lipopeptide is more effective in inducing CTL than the corresponding peptide. Conclusion: The capacity of the peptides and lipopeptides to up regulate class I MHC expression can be used as a prediction for their potential to function as a CTL epitope. So far we identified one CTL epitope on the 38 kD protein.

1P.4.11.18 1 Complete amino acid sequence of a novel 24 kD secreted mycobacterial lipoprotein relevant to cellular Immunity F. Oftung’, H.G. Wiker*, A. Deggerda13,K.E.A. Lundin4, A.S. Mustafa5. ’Dept. of VaccinologL:National institute of Public Health, Oslo, Norwax */GRI, Oslo, Norwax 4IT/, The Nationa/ Hospital, Oslo, Norway 3DYNAL AS, Oslo, Norwax 5Dept. of Microbio/logy:Faculty of Medicine, Kuwait University;Kuwait

Introduction: The objective of the study was to identify and characterize novel mycobacterial protein antigens with relevance to cellular immune responses and tuberculosis subunit vaccine design. Materlalsand Methods:Identification of a novel mycobacterial protein antigen was performed by screening of a genomic M. leprae Agtll DNA library by M. leprae reactive human CD4+ T cell clones as probes. DNA sequencing: dideoxy chain termination method. Tcell epitope and MHC restriction analysis (anti HLA blocking and panel studies) was based on synthetic peptides as antigen. Resulta: We present the complete amino acid sequence of a novel M. tuberculosis protein antigen common to M. leprae and the vaccine strain M. /bov/.sBCG with a deduced molecular weight of 24.1 kD. The 233 amino acid long reading frame contains a signal peptide sequence for secretion and a consensus motif for lipid conjugation, which suggest that the mature protein is a secreted antigen probably located to the mycobactetial cell wall as a lipoprotein. The complete gene was identified within the Sanger Centre data base (UK) by searching with a 123 bp C terminal DNA sequence isolated from a genomic M. /eprae Agtll library by expression of an epitope recognized by human T cell clones. The same T cell clones were used to confirm the deduced molecular mass in a T cell Western analysis as well as define the epitope recognized and its MHC restriction. Conclusion:We have identified and described the structure of a novel secreted mycobactetfal lipoprotein antigen with relevance to human cellular immunity and subunit vaccine design.

)P.4.11 .19( Local antibody responses to mycobacterlal antlgens in the lung of HIV-and HIV+ pulmonary tuberculosis patients K. Lyashchenko ‘, R.Condos 2, N. Schluger *, W. Ram*, ML. Gennaro I. ’Public Health Research Institute, New York,NY USA, *New York University Me&a/ Center. New York, NL: USA

Introduction:The lung is a major target for both HIV and ~cobactetium tuberculosis. Tuberculosis (TB) has become the leading cause of death for HIV-infected individuals. In the present study we characterized specific antibody responses to protein antigens of M. tubercu/osis in broncho-alveolar lavage (BAL) fluids of both HIV- and HIV+ patients with active pulmonary TB. Materlals and Mthods: The BAL fluids were obtained separately from radiologically involved and uninvolved lung lobes. In blind-coded expetfments, antibodies of different isotypes against secreted protein antigens of M. tubemu/asis were measured by ELISA using M. fubercukasisculture filtrates and eleven highly purfffed recombinant proteins of M. tubenx/osis. Reaufts: Our most significant findings are: 1) Specific antibodies against M. tuberculosis culture filtrates detected in TB+ BAL fluids are mostly IgG. Levels of IgA antibodies are very low, and IgM antibodies are not measurable by ELISA. 2) Levels oftheIgG antibodies were significantly higher in BALfluids obtained from involved lung lobes than from uninvolved sites in the same TB patients. 3) Levels of IgG antibodies in BAL fluids of HIV-l+ patients were comparable to those measured in HIV-l- TB patients. 4) IgG antibodies in BAL fluids from different patients all reacted predominantly with the same three purified mycobactetfal antigens. This was in contrast with IgG antibodies in sera from TB patients that usually recognized variable patterns of the protein antigens of M. tubercu/osis. Conoluslon:Our findings suggest that, in pulmonary TB, humoral immune responses in the lung involve primarily IgG antibodies specific to a limited number of secreted protein antigens of M. tuberculosis, and that these responses may only be marginally affected by HIV-co-infection. These observations offer a new tool to study the relationships between local and systemic immune responses in TB associated with HIV. Detection of BAL fluid antibodies to M. tubercu/osis antigens can be also used to monitor treatment course during TB-HIV-co-infection.

(P.4.11.20 ] MTC28, a novel immunodomlnant protein antigen speclflc to Mycobacfdum fuberculosiacomplex M.L. Gennaro, K. Lyashchenko. R. Colangeli, C. Manta. PuWic Health Research Institute, New York, NY USA Introduction: Protein antigens actively secreted by M. fuberculoss during infection are major targets for both B and T cell immune responses in tuberculosis (TB). Identification of new immunodominant secreted proteins of M. tubenxkxis is strongly needed for developing alternative vaccines against TB as well as for improving immunodiagnostfcs in the disease. Materialsand Methods:An M. tubenx/osis recombinant phage expression library was screened with a rabbit immune serum raised against whole culture filtrates of M. tubercu/csis. One positive clone was subjected to subcloning and nucleotide sequencing and found to encode a novel gene of M. tubercu/osis, m&28. The recombinant MTC28 was expressed in E. co/i and purified as a polyhistidine tagged fusion protein by three-step sequential chromatography. Immunologic properties of the putffied antigen were characterized in antibody and cell-mediated immune responses by ELISA and by skin tests in guinea pigs immunized with live M. twv/.s BCG or M. avium, or with killed M. tubercukxis in adjuvant. Rwultr: A novel gene that encodes a protein present in the culture filtrate of M. tuberculosis was cloned and sequenced. The gene named, m&28, encoded a mature protein of 278 amino acid residues preceded by a 32 amino acid secretion signal peptide. By genomic Southern blot analysis, the mtc.28gene was detected in mycobacterfa of the M. fu6ercu/o&complex but not in other mycobacterial species. Highly purified, endotoxin-free MTC28 protein reacted with the rabbit immune serum used for the library screening in both ELISA and Western blotting. In the guinea pig experiments, the MTC28 elicited strong antibody and skin test responses in all the animals immunized with M. bov/s BCG, but in none given M. avium or killed M. tuberculosis. Conclusion:Immunologic characterization of the MTC28 recently identified in our laboratory clearly indicates that this novel protein is a potent immunodominant antigen presumably secreted by M. tuberculosis and highly specific to the M. fuberculosiscomplex in both antibody and T cell responses during infection. lmmunodiagnostic significance of the MTC28 in human and/or bovine TB is currently under evaluation.