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Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of post-exposure or therapeutic tuberculosis vaccines

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(1)Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of post-exposure or therapeutic tuberculosis vaccines Lin, M.Y.. Citation Lin, M. Y. (2009, December 15). Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of postexposure or therapeutic tuberculosis vaccines. Retrieved from https://hdl.handle.net/1887/14507 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/14507. Note: To cite this publication please use the final published version (if applicable)..

(2)  Chapter3 IdentificationofCD4+andCD8+Tcellresponsesto MycobacteriumtuberculosisDosRregulon encodeddormancyantigensandmappingofHLA classIandIIrestrictedpeptideepitopes.         . ƒ›‘—‰‹ȗǡ͓ǡ”‹•–ƒǤ˜ƒ‡‹Œ‰ƒƒ”†‡ȗǡ͓ǡ‡‹‡‡ Ǥ ”‹‰‰‡ȗǡ͓ǡ‡‡•ǤǤǤ ”ƒ‡ȗǡ ͓ǡ—•ƒ Ǥ Ǥ˜ƒ†‡‡†‡ȗǡ͓ǡ‡‹‡‡ ‡Ž—ȗǡ͓ǡ ƒ‘—–‡””‹ŒˆŠ‘—–ȗǡƒ†”ƒ”‡†͓ǡ ‹ Š°ŽǤŽ‡‹͓ǡȗǡ‘ ǤǤ––‡Š‘ˆˆȗǡ͓  ͓‡’ƒ”–‡–‘ˆ

(3) ˆ‡ –‹‘—•‹•‡ƒ•‡•ƒ†ȗ‡’ƒ”–‡–‘ˆ

(4) —‘Š‡ƒ–‘Ž‘‰›ƬŽ‘‘† ”ƒ•ˆ—•‹‘ǡ‡‹†‡‹˜‡”•‹–›‡†‹ ƒŽ‡–”‡ǡ‡‹†‡ǡ–Š‡‡–Š‡”Žƒ†•Ǥ  Manuscriptinpreparation.

(5) Chapter3. Abstract ˆˆ‡ –‹˜‡ ‰Ž‘„ƒŽ ƒ–‹Ǧ–—„‡” —Ž‘•‹• ȋȌ •–”ƒ–‡‰‹‡• ‹˜‘Ž˜‡• †‡˜‡Ž‘’‡– ‘ˆ „‡––‡” ˜ƒ ‹‡•ǡ‹ Ž—†‹‰’”‡˜‡–‹˜‡ƒ•™‡ŽŽƒ•’‘•–Ǧ‡š’‘•—”‡Ǧ‘”–Š‡”ƒ’‡—–‹ Ǧ˜ƒ ‹‡•Ǥ Š‡ Žƒ––‡” ƒ‹ ƒ– ’”‡˜‡–‹‰  ”‡ƒ –‹˜ƒ–‹‘ ‹ ƒŽ”‡ƒ†› ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ•ǡ ’ƒ”–‹ —Žƒ”Ž›•‹ ‡–Š‡™‹†‡Ž›—•‡† Ǧ˜ƒ ‹‡ˆƒ‹Ž•–‘’”‘–‡ –ƒ‰ƒ‹•–”‡ƒ –‹˜ƒ–‹‘Ǥ ˜‡” ʹ „‹ŽŽ‹‘ ’‡‘’Ž‡ ƒ”‡ –Š‘—‰Š– –‘ „‡ Žƒ–‡–Ž› ‹ˆ‡ –‡† ™‹–Š Mycobacterium tuberculosis ȋM. tuberculosisȌǤ ƒ”‰‡– ƒ–‹‰‡• •—‹–ƒ„Ž‡ ˆ‘” ’‘•–Ǧ‡š’‘•—”‡ ˜ƒ ‹ƒ–‹‘ ‡‡†–‘„‡‡š’”‡••‡†„›M.tuberculosis†—”‹‰ Ž‹‹ ƒŽŽƒ–‡ ›ǡ‹Ǥ‡ǤŽƒ–‡•–ƒ‰‡‹ˆ‡ –‹‘Ǥ ‡ ’”‡˜‹‘—•Ž› ”‡’‘”–‡† Š—ƒ  ‡ŽŽ ƒ –‹˜‹–› ƒ‰ƒ‹•– M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡•ǡ ™Š‹ Š ƒ”‡ •–”‘‰Ž› —’Ǧ”‡‰—Žƒ–‡† †—”‹‰ –Š‡ M. tuberculosis •–”‡•• ”‡•’‘•‡—’‘‹–”ƒ ‡ŽŽ—Žƒ”‹ˆ‡ –‹‘Ǥ —ƒ ‡ŽŽ”‡•’‘•‡•–‘M.tuberculosis‘• ”‡‰—Ž‘‡ ‘†‡†ƒ–‹‰‡•™‡”‡ƒ••‘ ‹ƒ–‡†™‹–Š ‘–”‘Ž‘ˆŽƒ–‡–‹ˆ‡ –‹‘ǡ„—––Š‡ ƒ–—”‡ ‘ˆ –Š‡ ”‡•’‘•‡ ”‡ƒ‹• —‹†‡–‹ˆ‹‡†Ǥ ”‘–‡ –‹˜‡ ‡ŽŽ—Žƒ” ‹—‹–› –‘ M. tuberculosis –Š”‹˜‡• ‘ „‘–Š ͶΪ ƒ† ͺΪ  ‡ŽŽ ‡†‹ƒ–‡† ”‡•’‘•‡•Ǥ ‡”‡ǡ ™‡ ‹†‡–‹ˆ› –Š‡ ’”‡•‡ ‡ ‘ˆ „‘–Š ͶΪ ƒ† ͺΪ  ‡ŽŽ ”‡•’‘•‡• –‘™ƒ”†• ƒ •‡”‹‡• ‘ˆ ‹—‘†‘‹ƒ– M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ‹ M. tuberculosis ”‡•’‘•‹˜‡ ‹†‹˜‹†—ƒŽ•Ǥ ‡ ˆ—”–Š‡” •Š‘™ –Šƒ– M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ‘–ƒ‹ „‘–Š ͶΪ ƒ† ͺΪ  ‡ŽŽ ‡’‹–‘’‡•ǡ ™Š‹ Š ƒ”‡ ”‡ ‘‰‹œ‡† ‹ –Š‡ ‘–‡š–‘ˆ Ǧ Žƒ••

(6)

(7) ‘” Žƒ••

(8) ‘Ž‡ —Ž‡•ǡ”‡•’‡ –‹˜‡Ž›Ǥ”‘Ž‹ˆ‡”ƒ–‹˜‡”‡•’‘•‡•™‡”‡ ˜‹•—ƒŽ‹œ‡†—•‹‰ƒ „ƒ•‡†ƒ••ƒ›ǡƒŽŽ‘™‹‰•‹—Ž–ƒ‡‘—•†‡–‡ –‹‘‘ˆͶΪƒ•™‡ŽŽ ƒ•ͺΪ’”‘Ž‹ˆ‡”ƒ–‹˜‡ ‡ŽŽ”‡•’‘•‡•–‘–Š‡•ƒ‡ƒ–‹‰‡•ƒ†’‡’–‹†‡•ǤŠ‡•‡”‡•—Ž–• •‹‰‹ˆ‹ ƒ–Ž› ‹ ”‡ƒ•‡ ‘—” —†‡”•–ƒ†‹‰ ‘ˆ –Š‡ Š—ƒ ‹—‡ ”‡•’‘•‡ –‘ M. tuberculosis ’Šƒ•‡Ȁ•–ƒ‰‡•’‡ ‹ˆ‹ ƒ–‹‰‡•ǡƒ† ƒ› Š‡Ž’ ‹ †‡•‹‰‹‰M. tuberculosis ‘•”‡‰—Ž‘‡ ‘†‡†ƒ–‹‰‡‘”’‡’–‹†‡„ƒ•‡†˜ƒ ‹ƒ–‹‘ƒ’’”‘ƒ Š‡•–‘Ǥ . ͷͺ.

(9) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. Introduction —ƒŽŽ›ǡ Žƒ‹•‘˜‡”ͳǤͷ‹ŽŽ‹‘Ž‹˜‡•ƒ† ƒ—•‡•ƒ –‹˜‡ȋ‹ˆ‡ –‹‘—•Ȍ†‹•‡ƒ•‡‹ ‘˜‡”ͻ‹ŽŽ‹‘ ƒ•‡•Ǥ

(10) –‹•‡•–‹ƒ–‡†–Šƒ–‘˜‡”ʹ„‹ŽŽ‹‘’‡‘’Ž‡ƒ”‡Žƒ–‡–Ž›‹ˆ‡ –‡†™‹–Š MycobacteriumtuberculosisȋM.tuberculosisȌǡ–Š‡ ƒ—•ƒ–‹˜‡ƒ‰‡–‘ˆ–—„‡” —Ž‘•‹•ȋȌǤ Š‹• ˜ƒ•– ”‡•‡”˜‘‹” ‘ˆ Žƒ–‡–Ž› ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ• ˆ‘”• ƒ ƒŒ‘” •‘—” ‡ ‘ˆ ‡™  ƒ•‡•Ǥ ‡ ‹ ‡˜‡”› ͳͲ M. tuberculosis ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ• ™‹ŽŽ ‡˜‡–—ƒŽŽ› †‡˜‡Ž‘’ ƒ –‹˜‡  †—”‹‰ –Š‡‹” Ž‹ˆ‡–‹‡ ™Š‡”‡ƒ• –Š‡ ”‡ƒ‹†‡” ‹• ƒ„Ž‡ –‘ ‘–ƒ‹ –Š‡ „ƒ ‹ŽŽ‹ ™‹–Š‘—– †‡˜‡Ž‘’‹‰ ƒ› Ž‹‹ ƒŽ •›’–‘•Ǥ —””‡– ‘–”‘Ž ‘ˆ –Š‡  ’ƒ†‡‹  ‹• •‡”‹‘—•Ž›‹’‡†‡†„›–Š‡•–”‘‰‹ ”‡ƒ•‡‹‘”„‹†‹–›ƒ†‘”–ƒŽ‹–›†—”‹‰ƒ –‹˜‡

(11)  ‘Ǧ‹ˆ‡ –‹‘ǡ ƒ† –Š‡ ‘–‹—‘—• ”‹•‡ ‘ˆ —Ž–‹Ǧ†”—‰ ”‡•‹•–ƒ– ȋȌ ƒ† ‡š–‡•‹˜‡Ž›†”—‰Ǧ”‡•‹•–ƒ–ȋȌM.tuberculosis•–”ƒ‹•ȋ͵ͺȌǤ Mycobacterium bovis ƒ ‹ŽŽ—• ƒŽ‡––‡Ǧ —±”‹ ȋ Ȍ ‹• –‘†ƒ›ǯ• ‘Ž› ƒ˜ƒ‹Žƒ„Ž‡  ˜ƒ ‹‡Ǥ   ’”‘–‡ –• ƒ‰ƒ‹•– •‡˜‡”‡ ˆ‘”• ‘ˆ  ‹ ›‘—‰ Š‹Ž†”‡ ƒ† ƒ‰ƒ‹•– Ž‡’”‘•›ǡ „—– †‘‡• ‘– ‡ˆˆ‹ ‹‡–Ž› ƒ† ‘•‹•–‡–Ž› ’”‘–‡ – ƒ‰ƒ‹•– ’—Ž‘ƒ”›  ‹ ƒ†—Ž–•ȋͺǡͳ͵ȌǤˆˆ‡ –‹˜‡ƒ–‹Ǧ•–”ƒ–‡‰‹‡•‘–‘Ž›”‡“—‹”‡„‡––‡”’”‡˜‡–‹˜‡˜ƒ ‹‡•ǡ „—– ƒŽ•‘ ˜ƒ ‹‡• ‡š‡”–‹‰ ’‘•–Ǧ‡š’‘•—”‡Ȁ–Š‡”ƒ’‡—–‹  ƒ –‹˜‹–› ȋʹͷǡͶͲȌ •‹ ‡   ‹• ‹‡ˆˆ‡ –‹˜‡ ‹ Žƒ–‡–Ž› ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ•Ǥ ‘•–Ǧ‡š’‘•—”‡  ˜ƒ ‹‡• •Š‘—Ž† ƒ‹ ƒ– ’”‡˜‡–‹‰ ”‡ƒ –‹˜ƒ–‹‘ ‘ˆ  ‹ˆ‡ –‹‘ ‹ Žƒ–‡–Ž› ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ• „› ‹†— ‹‰ ”‘„—•– ‹—‹–› –‘ ƒ–‹‰‡• –Šƒ– ƒ”‡ ‡š’”‡••‡† „› ’‡”•‹•–‹‰ M. tuberculosis „ƒ ‹ŽŽ‹ †—”‹‰ Žƒ–‡– ‹ˆ‡ –‹‘Ǥ — Š ‹—‡ ”‡•’‘•‡• ƒ”‡ ‘•‹†‡”‡† ‡••‡–‹ƒŽ ‹ ‘–”‘ŽŽ‹‰ǡ‘”‡˜‡„‡––‡”ǡ‡”ƒ†‹ ƒ–‹‰’‡”•‹•–‹‰„ƒ ‹ŽŽ‹ǤŠ‹•Ž‹‡Ž›”‡“—‹”‡•‹†— –‹‘ ‘ˆ ƒ†‡“—ƒ–‡ ͶΪ ƒ† ͺΪ Ǧ ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– –Š‡•‡ †‘”ƒ › ƒ••‘ ‹ƒ–‡† M. tuberculosis ƒ–‹‰‡•Ǥ ‡ ‡–Ž›ǡ ™‡ •Š‘™‡† –Šƒ– ‰‡‡• ‘ˆ –Š‡ M. tuberculosis ‘• ȋ˜͵ͳ͵͵ Ȍ”‡‰—Ž‘‡ ‘†‡ƒ–‹‰‡•–Šƒ– ƒ‹†— ‡•‹‰‹ˆ‹ ƒ– ‡ŽŽ”‡•’‘•‡•‹M. tuberculosis‹ˆ‡ –‡†‹†‹˜‹†—ƒŽ•ȋŽƒ–‡–Ž›‘”ƒ –‹˜‡Ž›‹ˆ‡ –‡†ȌȋͳͺȌǤŠ‡ͶͺǦ‰‡‡‘• ”‡‰—Ž‘‹•‡š’”‡••‡†„›–—„‡” Ž‡„ƒ ‹ŽŽ‹†—”‹‰‹˜‹–”‘‡š’‘•—”‡–‘Š›’‘š‹ƒǡŽ‘™†‘•‡ ‹–”‹  ‘š‹†‡ ƒ† ƒ”„‘ ‘‘š‹†‡ǡ ‘†‹–‹‘• –Š‘—‰Š– –‘ „‡ ‡ ‘—–‡”‡† „› M. tuberculosis‹˜‹˜‘™Š‡’‡”•‹•–‹‰‹‹—‘Ǧ ‘’‡–‡–Š‘•–•ȋ͵͸ȌǤ’’”‘š‹ƒ–‡Ž› ŠƒŽˆ ‘ˆ –Š‡ M. tuberculosis ‘• ”‡‰—Ž‘ ‹• ƒŽ•‘ ‡š’”‡••‡† ‘˜‡” ’”‘Ž‘‰‡† ’‡”‹‘†• ‘ˆ –‹‡ ‹ –Š‡ ”‡ ‡–Ž› ’”‘’‘•‡† ‡†—”‹‰ Š›’‘š‹ƒ ”‡•’‘•‡ ȋ Ȍ ‘†‡Ž ȋ͵ͲȌǤ

(12) —‹–› –‘ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• •‡‡• –‘ „‡ ƒ••‘ ‹ƒ–‡† ™‹–Š ‘–”‘Ž‘ˆŽƒ–‡–‹ˆ‡ –‹‘ǡ•‹ ‡•‡˜‡”ƒŽ‘•”‡‰—Ž‘‡ ‘†‡†ƒ–‹‰‡•™‡”‡ ȋ’”‡ˆ‡”‡–‹ƒŽŽ›Ȍ”‡ ‘‰‹œ‡†„›‹†‹˜‹†—ƒŽ•™‹–ŠŽƒ–‡–‹ˆ‡ –‹‘ȋ

(13) ȌȋͳͲǡͳͺǡʹͻȌǤ ‡–Š‡”‡ˆ‘”‡Š›’‘–Š‡•‹œ‡†–Šƒ–‹—‡”‡•’‘•‡•–‘–Š‡•‡ƒ–‹‰‡•‹‰Š– ‘–”‹„—–‡ –‘–Š‡ ‘–”‘Ž‘ˆ’‡”•‹•–‡–M.tuberculosis‹ˆ‡ –‹‘Ǥ ‘™‡˜‡”ǡ –Š‡ ‡šƒ – ƒ–—”‡ ‘ˆ –Š‡•‡ Š—ƒ  ‡ŽŽ ”‡•’‘•‡• –‘ M. tuberculosis ‘• ”‡‰—Ž‘‡ ‘†‡†ƒ–‹‰‡•Šƒ•‘–„‡‡•–—†‹‡†‹†‡–ƒ‹ŽǤ‘–ŠͶΪƒ†ͺΪ ‡ŽŽ• ’ƒ”–‹ ‹’ƒ–‡‹–Š‡‘’–‹ƒŽ’”‘–‡ –‹˜‡”‡•’‘•‡–‘M.tuberculosisǤ ‡”‡ǡ™‡†‡• ”‹„‡ƒ —„‡” ‘ˆ ”‡Ž‡˜ƒ– ƒ† Š‹‰ŠŽ› ”‡ ‘‰‹œ‡† M. tuberculosis ‘• ƒ–‹‰‡• –Šƒ– ‹†— ‡ „‘–Š ͶΪ ƒ† ͺΪ Š—ƒ  ‡ŽŽ ”‡•’‘•‡• ‹ › ‘„ƒ –‡”‹ƒ ’”‹‡† ‹†‹˜‹†—ƒŽ•Ǥ ‡•’‘•‡•™‡”‡ƒ’’‡†–‘ƒ•‡”‹‡•‘ˆM.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡†’‡’–‹†‡•ǡ ™Š‹ Š ™‡”‡ ”‡ ‘‰‹œ‡† ‹ –Š‡ ‘–‡š– ‘ˆ  Žƒ••

(14)  ƒ†  Žƒ••

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(16)  ‘Ž‡ —Ž‡•Ǥ ‡–ƒ‹Ž‡† ‡’‹–‘’‡ ƒ’’‹‰ ‘ˆˆ‡”• –Š‡ ’‘–‡–‹ƒŽ –‘ ‡Ž— ‹†ƒ–‡ ’‘••‹„Ž‡ •—„†‘‹ƒ– ‡’‹–‘’‡•Ǣ –Š‡•‡ ‹‰Š– „‡ ‡š’Ž‘‹–‡† ‹ †‡•‹‰‹‰ ‹’”‘˜‡† ˜ƒ ‹‡• „› ‹†— ‹‰. ͷͻ.

(17) Chapter3. ‹—‹–› –‘ ƒ „”‘ƒ†‡” ‡’‹–‘’‡ ”‡’‡”–‘‹”‡ –Šƒ ™‘—Ž† „‡ •‡‡ ˆ‘ŽŽ‘™‹‰ ƒ–—”ƒŽ ‹ˆ‡ –‹‘‘”’”‘–‡‹˜ƒ ‹ƒ–‹‘ȋʹʹǡʹͶȌǤ.  MaterialsandMethods Studysubjects —ˆˆ› ‘ƒ–•ˆ”‘–™‡–›Ǧ‘‡‹˜‹–”‘”‡•’‘•‹˜‡ȋΪȌ Š‡ƒŽ–Š›ƒ‘›‘—•ǡ  –›’‡† „Ž‘‘† „ƒ †‘‘”• ™‡”‡ ‹ Ž—†‡† ‹ –Š‹• •–—†›Ǥ ‘ ’”‹‘” •‡Ž‡ –‹‘ ™ƒ• ƒ†‡ ‘ ‡”‹‰  –›’‡Ǥ ‘ Ž‹‹ ƒŽ ‹ˆ‘”ƒ–‹‘ ‹• ƒ˜ƒ‹Žƒ„Ž‡ ˆ”‘ –Š‡•‡ ƒ‘›‘—• Š‡ƒŽ–Š› †‘‘”• ‘ ‡”‹‰  ‹ˆ‡ –‹‘Ȁ‡š’‘•—”‡ǡ  ‘–ƒ – Š‹•–‘”› ‘”   ˜ƒ ‹ƒ–‹‘ǡ ‡š ‡’– –Šƒ– –Š‡› ™‡”‡ Š‡ƒŽ–Š› ƒ† Šƒ† ‘ Š”‘‹  ˜‹”ƒŽ ‹ˆ‡ –‹‘•Ǥ ‘™‡˜‡”ǡ ‰‹˜‡ –Š‡ Ž‘™  ‹ ‹†‡ ‡ ‹ –Š‡ ‡–Š‡”Žƒ†•ǡ ƒ† –Š‡ Žƒ  ‘ˆ ƒ ƒ–‹‘ƒŽ   ˜ƒ ‹ƒ–‹‘ ’‘Ž‹ ›ǡ ‘‡ ‘ˆ –Š‡•‡ ’ƒ”ƒ‡–‡”• ™‘—Ž† „‡ ‡š’‡ –‡† –‘ „‡ ‘ˆ Š‹‰Š •‹‰‹ˆ‹ ƒ ‡ ‹ –Š‹• ‘Š‘”–Ǥ ††‹–‹‘ƒŽŽ›ǡ  ˆ”‘ ͷ  ’ƒ–‹‡–• ƒ† ͺ –—„‡” —Ž‹ •‹–‡•–ȋȌ ‘˜‡”–‡”•™‡”‡•–—†‹‡†ǤŽ‘‘†‘ˆ–Š‡•‡ͳ͵†‘‘”•™‡”‡‘„–ƒ‹‡†„› ˜‡ƒ’— –—”‡ ƒˆ–‡” ™”‹––‡ ‹ˆ‘”‡† ‘•‡– ™ƒ• ‘„–ƒ‹‡†Ǥ Š‡ •–—†› ’”‘–‘ ‘Ž ™ƒ• ƒ’’”‘˜‡† „›–Š‡

(18) •–‹–—–‹‘ƒŽ ‡˜‹‡™ ‘ƒ”†‘ˆ–Š‡ ‡‹†‡ ‹˜‡”•‹–› ‡†‹ ƒŽ ‡–”‡ ȋȌǤ™‡”‡‹•‘Žƒ–‡†—•‹‰ ‹ ‘ŽŽ†‡•‹–›‰”ƒ†‹‡– ‡–”‹ˆ—‰ƒ–‹‘ƒ†•–‘”‡†‹ Ž‹“—‹†‹–”‘‰‡—–‹Ž—•‡Ǥ  M.tuberculosisantigensandpeptides ‡ ‘„‹ƒ– ’”‘–‡‹• ™‡”‡ ’”‘†— ‡† ƒ• †‡• ”‹„‡† ’”‡˜‹‘—•Ž› ȋͳͶȌǤ ”‹‡ˆŽ›ǡ •‡Ž‡ –‡† M. tuberculosis ͵͹˜ ‰‡‡• ™‡”‡ ƒ’Ž‹ˆ‹‡† „›  ˆ”‘ ‰‡‘‹  ͵͹˜  ƒ† Ž‘‡† „› ƒ–‡™ƒ› ‡ Š‘Ž‘‰› ȋ

(19) ˜‹–”‘‰‡ǡ ƒ ‹‡‰‘ǡ ȌǤ ”‘–‡‹• ™‡”‡ ‘˜‡”Ǧ ‡š’”‡••‡† ‹ Escherichia coli •–”ƒ‹ ʹͳȋ͵Ȍ ƒ† ’—”‹ˆ‹‡† ƒ• †‡• ”‹„‡† ȋͳͶȌǤ ŽŽ ”‡ ‘„‹ƒ–’”‘–‡‹•™‡”‡•—„Œ‡ –‡†–‘“—ƒŽ‹–› ‘–”‘Žƒ••ƒ›•‹ Ž—†‹‰•‡“—‡ ‹‰ǡ •‹œ‡ ƒ† ’—”‹–› Š‡ ǡ ”‡•‹†—ƒŽ ‡†‘–‘š‹ Ž‡˜‡Ž•ǡ ‘Ǧ•’‡ ‹ˆ‹  Ǧ ‡ŽŽ •–‹—Žƒ–‹‘ ƒ† ’‘–‡–‹ƒŽ ‡ŽŽ—Žƒ” –‘š‹ ‹–› ȋͳͻȌǤ —”‹ˆ‹‡† ’”‘–‡‹ †‡”‹˜ƒ–‹˜‡ ȋǡ „ƒ– Š ͶͻȌ ™ƒ• ’—” Šƒ•‡†ˆ”‘–ƒ–‡•‡”—

(20) •–‹–—–‡ǡ‡ƒ”Ǥ ›–Š‡–‹  ’‡’–‹†‡• ™‡”‡ •›–Š‡•‹œ‡† ƒ• ’”‡˜‹‘—•Ž› †‡• ”‹„‡† ȋͳ͹ȌǤ ‡’–‹†‡• ˆ”‘ M. tuberculosis ‘• ƒ–‹‰‡• ˜ͳ͹͵͵ ǡ ˜ʹͲʹͻ ǡ ˜ʹͲ͵ͳ ǡ ˜ʹ͸ʹ͹  ƒ† ”‡ˆ‡”‡ ‡ ƒ–‹‰‡ ‰ͺͷ ™‡”‡ ʹͲǦ‡”• ’‡’–‹†‡• ™‹–Š ͳͲ ƒ‹‘ ƒ ‹†• ȋƒƒȌ ‘˜‡”Žƒ’ ‡š ‡’– ’‡’–‹†‡• ʹͲǦʹʹ ‘ˆ ‰ͺͷ ™Š‹ Š ™‡”‡ ͳͷǦ‡”• ™‹–Š ͳͲ ƒƒ ‘˜‡”Žƒ’Ǥ Š‡ ʹͲǦ‡” ’‡’–‹†‡• ‘ˆ ˜ͳ͹͵͵ ǡ ˜ʹͲʹͻ  ƒ† ˜ʹ͸ʹ͹  ™‡”‡ ‡Ž‘‰ƒ–‡† ™‹–Š –™‘ Ž›•‹‡ ȋȌ ”‡•‹†—‡•ƒ––Š‡Ǧ–‡”‹ƒŽ–‘‹’”‘˜‡•‘Ž—„‹Ž‹–›Ǥƒ†‹†ƒ–‡ Ǧʹ‡’‹–‘’‡• ‘•‹•–‡† ‘ˆ ͻǦ‡”• ƒ† ƒ†‹†ƒ–‡ Ǧ͵ ‡’‹–‘’‡• ™‡”‡ ͳͷ ‘” ͳ͸Ǧ‡”•Ǥ Š‡ ǦȗͲʹͲͳǦ ”‡•–”‹ –‡†ǡ

(21) Ǧͳ’ͳ͹ ƒ‰͹͹Ȃͺͷ‡’‹–‘’‡ȋȌ™ƒ•—•‡†ƒ• ‘–”‘Ž’‡’–‹†‡ȋ͵ͶȌǤ  GenerationofCD4+Tcelllines  ‡ŽŽŽ‹‡•™‡”‡‰‡‡”ƒ–‡†ƒ•’”‡˜‹‘—•Ž›†‡• ”‹„‡†ȋʹȌǤ

(22) „”‹‡ˆǡ™‡”‡‹ —„ƒ–‡† ƒ–ͳǦʹšͳͲ͸ ‡ŽŽ•Ȁ™‡ŽŽ‹ʹͶǦ™‡ŽŽ’Žƒ–‡•ȋ— ǡ‘•‹Ž†‡ǡ‡ƒ”Ȍ‹–Š‡ ’”‡•‡ ‡‘ˆ M. tuberculosis Ž›•ƒ–‡ ȋͷ P‰ȀŽȌ ‹

(23)  •—’’Ž‡‡–‡† ™‹–Š ͷͲ ȀŽ ’‡‹ ‹ŽŽ‹ǡ ͷͲ P‰ȀŽ •–”‡’–‘› ‹ ȋ

(24) ˜‹–”‘‰‡ǡ ”‡†ƒǡ Š‡ ‡–Š‡”Žƒ†•Ȍ ƒ† ͳͲΨ ’‘‘Ž‡† Š—ƒ •‡”—Ǣ ˆ—”–Š‡” ƒ††”‡••‡† ƒ• —Ž–—”‡ ‡†‹—Ǥ ˆ–‡” ͸ †ƒ›•ǡ

(25) Ǧʹ ȋʹͷ ȀŽȌ ȋ‡–—•ǡ ͸Ͳ.

(26) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. ‡”›˜‹ŽŽ‡ǡǡȌ™ƒ•ƒ††‡†ƒ† —Ž–—”‡•™‡”‡ ‘–‹—‡†ˆ‘”ƒ‘–Š‡”ʹ–‘͵™‡‡• ‹’”‡•‡ ‡‘ˆ

(27) ǦʹǤǦ ‡ŽŽ•™‡”‡ˆ”‘œ‡ƒ†•–‘”‡†‹Ž‹“—‹†‹–”‘‰‡—–‹Ž—•‡Ǥ   GenerationofCD8+Tcelllines ͺΪ ‡ŽŽŽ‹‡•™‡”‡‰‡‡”ƒ–‡†„›—•‹‰ȋƒ—–‘Ž‘‰‘—•Ȍ†‡†”‹–‹  ‡ŽŽ•ȋȌƒ•ƒ–‹‰‡ ’”‡•‡–‹‰ ‡ŽŽȋȌƒ†–Š”‡‡M.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡† ƒ†‹†ƒ–‡ Ǧ ʹ‡’‹–‘’‡•Ǥˆ”‘ƒ ǦʹΪ’ƒ–‹‡–ȋ Ǧʹǡ͵ǡƒ†ͳͳȌ™‡”‡—•‡†ǣƒ– †ƒ›Ǧ͹ǡͳͶΪ ‡ŽŽ•™‡”‡‹•‘Žƒ–‡†„›—•‹‰„‡ƒ†•ȋ‹Ž–‡›‹ǡ ‡”ƒ›Ȍƒ†’Žƒ–‡† ‹͸Ǧ™‡ŽŽ•’Žƒ–‡•ƒ–͵šͳͲ͸ ͳͶΪ ‡ŽŽ•‹

(28) ǡͳͲΨ ǡ ‘–ƒ‹‹‰ͺͲ‰ȀŽ Ǧ  ȋ‹‘•‘—” ‡Ȍ ƒ† ͷͲͲ Ȁ Ž ”

(29) ǦͶ ȋ‡’”‘–‡ ŠȌ ˆ—”–Š‡” ”‡ˆ‡””‡† –‘ ƒ•  —Ž–—”‡ ‡†‹—ǡ ƒ– ͵͹‘ ƒ† ͷΨ ʹ –‘ ‹†— ‡ ‹ƒ–—”‡ • ȋ‹Ȍ ȋ͵ͷȌǤ ͺΪ  ‡ŽŽ• ™‡”‡ ‹•‘Žƒ–‡†ˆ”‘–Š‡ͳͶǦˆ”ƒ –‹‘Ǥ‘–ŠͺΪƒ†ͺǦˆ”ƒ –‹‘™‡”‡ˆ”‘œ‡ƒ†•–‘”‡† ‹ Ž‹“—‹† ‹–”‘‰‡ —–‹Ž —•‡Ǥ ‹• ™‡”‡ ƒ–—”‡† ‘ †ƒ› Ǧͳ ‹  —Ž–—”‡ ‡†‹—ǡ •—’’Ž‡‡–‡†™‹–ŠͷͲ‰ȀŽǤ–†ƒ›Ͳǡƒ–—”‡•ȋ•Ȍ™‡”‡Šƒ”˜‡•–‡†ƒ† ”‡•—•’‡†‡†‹

(30) ™‹–ŠͳΨ’‘‘Ž‡†Š—ƒ•‡”—‹–Š‡’”‡•‡ ‡‘ˆʹͷP‰ȀŽ’‡” Ǧʹ ƒ†‹†ƒ–‡‡’‹–‘’‡ƒ†͵P‰ȀŽEʹǦ‹ ”‘‰Ž‘„—Ž‹ȋ‹‰ƒǦŽ†”‹ Šǡ™‹Œ†”‡ Š–ǡ Š‡‡–Š‡”Žƒ†•Ȍˆ‘”ͶŠ‘—”•ƒ–͵͹‘ƒ†ͷΨʹ™‹–ŠŠ‘—”Ž›”‡•—•’‡•‹‘‘ˆ ‡ŽŽ•Ǥ ˆ–‡”™ƒ•Š‹‰ǡ’‡’–‹†‡Ž‘ƒ†‡†•™‡”‡‹””ƒ†‹ƒ–‡†ƒ–ʹͷͲͲ”ƒ†ǤͺΪ ƒ†ͺǦ  ‡ŽŽ ˆ”ƒ –‹‘•™‡”‡–Šƒ™‡†ƒ†‹š‡†‹ƒͳͲǣͳ”ƒ–‹‘ƒ†”‡•—•’‡†‡†ƒ–͵šͳͲ͸ ‡ŽŽ•‹ͳ Ž‹ƒʹͶǦ™‡ŽŽ•’Žƒ–‡ǡˆ‘”‹‰–Š‡”‡•’‘†‹‰’‘’—Žƒ–‹‘Ǥ‘–Š‡”‡•’‘†‡”• ‡ŽŽ•ͳ Ž •–‹—Žƒ–‹‘ —Ž–—”‡ ȋ‹””ƒ†‹ƒ–‡†ǡ ’‡’–‹†‡ ’—Ž•‡† •Ȍ ™ƒ• ƒ††‡† ȋ‹ ƒ ”ƒ–‹‘ ͳͲǣͳ ”‡•’‡ –‹˜‡Ž›Ȍ‹ —Ž–—”‡‡†‹—•—’’Ž‡‡–‡†™‹–ŠͳͲ‰ȀŽ

(31) Ǧ͹ƒ†ͷͲ’‰ȀŽ

(32) Ǧ ͳʹǤ – †ƒ› ͹ǡ ͳ Ž ‡†‹— ™ƒ• ”‡ˆ”‡•Š‡† ™‹–Š —Ž–—”‡ ‡†‹— ƒ†

(33) Ǧ͹ ƒ†

(34) Ǧʹ ‹ ˆ‹ƒŽ ‘ ‡–”ƒ–‹‘‘ˆʹͲ‰ȀŽƒ†ͷͲȀŽ”‡•’‡ –‹˜‡Ž›Ǥ–†ƒ›ͳʹǡͶšͳͲ͸ƒ—–‘Ž‘‰‘—•  ™‡”‡ •‡‡†‡† ƒ– ͶšͳͲ͸ ‡ŽŽ•ȀŽ ‹

(35)  ‹ ƒ ʹͶǦ™‡ŽŽ• ’Žƒ–‡Ǥ ˆ–‡” –™‘ Š‘—”•ǡ ‘Ǧƒ†Š‡”‡– ‡ŽŽ• ™‡”‡ ™ƒ•Š‡† ƒ™ƒ› ƒ† ʹͲ P‰ȀŽ ’‡” Ǧʹ ƒ†‹†ƒ–‡ ‡’‹–‘’‡ ƒ†͵P‰ȀŽEʹǦ‹ ”‘‰Ž‘„—Ž‹™‡”‡ƒ††‡†ˆ‘”ƒƒ††‹–‹‘ƒŽͶŠ‘—”•‹ƒ˜‘Ž—‡‘ˆͲǤͷ ŽȀ™‡ŽŽǤ Š‡ ”‡•’‘†‡”  —Ž–—”‡ ™ƒ• ”‡•—•’‡†‡† ƒ– ͳǤͷšͳͲ͸ ‡ŽŽ•ȀŽ ‹ —Ž–—”‡ ‡†‹—Ǥ‡†‹—™ƒ•”‡‘˜‡†ˆ”‘–Š‡’‡’–‹†‡’—Ž•‡†ƒ†Š‡”‡– ‡ŽŽ•ƒ†ͳŽ —Ž–—”‡ ™ƒ• ƒ††‡†Ǥ – †ƒ› ͳͶǡ —Ž–—”‡ ‡†‹— ™ƒ• ƒ††‡† ƒ†

(36) Ǧ͹ ƒ†

(37) Ǧʹ ‹ ˆ‹ƒŽ ‘ ‡–”ƒ–‹‘ ‘ˆ ʹͲ ‰ȀŽ ƒ† ͷͲ ȀŽ ”‡•’‡ –‹˜‡Ž›Ǥ Š‡  —Ž–—”‡ ™ƒ• ”‡•–‹—Žƒ–‡†‡˜‡”›͹Ǧͺ†ƒ›•ƒ•†‡• ”‹„‡†ƒ„‘˜‡ǡ–™‘†ƒ›•ˆ‘ŽŽ‘™‹‰”‡•–‹—Žƒ–‹‘ǡ

(38) Ǧ ͹ƒ†

(39) Ǧʹ™‡”‡ƒ††‡†ƒ•†‡• ”‹„‡†ƒ„‘˜‡Ǥ  Lymphocytestimulationassays ProliferationbasedonCFSEdilution: ™‡”‡–Šƒ™‡†ǡ™ƒ•Š‡†ƒ†Žƒ„‡Ž‡†ƒ–ͳͲ͹ ‡ŽŽ•ȀŽ™‹–Šͷρ ƒ”„‘š›ˆŽ—‘”‡• ‡‹ †‹ƒ ‡–ƒ–‡•— ‹‹‹†›Ž‡•–‡”ȋ Ǣ‘Ž‡ —Žƒ””‘„‡•ǡ‡‹†‡ǡŠ‡‡–Š‡”Žƒ†•Ȍˆ‘”ͳͲ ‹—–‡• ƒ– ͵͹ιǡ †ƒ” ‹ ȀͲǤͷΨ „‘˜‹‡ •‡”— ƒŽ„—‹ ȋǡ ‹‰ƒǦŽ†”‹ Šǡ ™‹Œ†”‡ Š–ǡ Š‡ ‡–Š‡”Žƒ†•ȌǤ ƒ„‡ŽŽ‹‰ ™ƒ• •–‘’’‡† „› ƒ††‹‰ ͳͲΨ Š‡ƒ–Ǧ ‹ƒ –‹˜ƒ–‡† ˆ‡–ƒŽ ƒŽˆ •‡”— ȋ Ǣ ”‡‹‡”ǡ Ž’Š‡ ƒȀ† ‹Œǡ Š‡ ‡–Š‡”Žƒ†•ȌǤ ‡ŽŽ• ™‡”‡™ƒ•Š‡†‹ȀͲǤͷΨƒ†”‡•—•’‡†‡†‹ —Ž–—”‡‡†‹—Ǥ‡ŽŽ•™‡”‡ƒ††‡† –‘ͻ͸Ǧ™‡ŽŽǦ„‘––‘‹ ”‘–‹–‡”’Žƒ–‡•ȋ— Ȍƒ–ͳǤͷšͳͲͷ ‡ŽŽ•Ȁ™‡ŽŽƒ†•–‹—Žƒ–‡†‹ †—’Ž‹ ƒ–‡ ™‹–Š ƒ–‹‰‡• ƒ– ͵͹‘ ƒ† ͷΨ ʹǤ –‹‰‡• ™‡”‡ –‡•–‡† ‹ –Š‡ ˆ‘ŽŽ‘™‹‰ ‘ ‡–”ƒ–‹‘•ǣ ‘• ƒ–‹‰‡•ǡ ‰ͺͷ ƒ† –Š‡‹” ”‡•’‡ –‹˜‡ •‹‰Ž‡ ’‡’–‹†‡•ǡ ‘–”‘Ž ’‡’–‹†‡ ȋ

(40) Ǧͳ ’ͳ͹ ƒ‰͹͹ȂͺͷȌǣ ƒŽŽ ƒ– ͳͲ P‰ȀŽǢ  ƒ– ͷ P‰ȀŽǢ ’Š›–‘Šƒ‡ƒ‰‰Ž—–‹‹. ͸ͳ.

(41) Chapter3. ȋ ǡ‡‡ŽǡȌƒ–ʹP‰ȀŽȋ’‘•‹–‹˜‡ ‘–”‘ŽȌǡƒ†‡†‹—‘Ž›ƒ•‡‰ƒ–‹˜‡ ‘–”‘ŽǤ ‡ŽŽ•‘ˆ†—’Ž‹ ƒ–‡•™‡”‡’‘‘Ž‡†ƒ†™ƒ•Š‡†™‹–Š™‹–ŠͲǤͳΨˆ‘”‡˜ƒŽ—ƒ–‹‘„› ˆŽ‘™ ›–‘‡–”›Ǥ ‹‹Žƒ” ’”‘Ž‹ˆ‡”ƒ–‹‘ ƒ† ”‡ƒ†‘—– ƒ••ƒ›• ™‡”‡ ’‡”ˆ‘”‡† ˆ‘” –Š‡ M. tuberculosis Ǧʹ ƒ†‹†ƒ–‡‡’‹–‘’‡•„—–™‹–Š‹‘” Šƒ‰‡•ǣ ‡ŽŽ•™‡”‡–‡•–‡†ƒ– ͳšͳͲͷ ‡ŽŽ•Ȁ™‡ŽŽ ȋ•‹šǦˆ‘Ž†Ȍ ‹ —Ž–—”‡ ‡†‹— ˆ‘” ‡‹–Š‡” •‹š ‘” –‡ †ƒ›•Ǥ – †ƒ› •‹šǡ •—’‡”ƒ–ƒ–• ™‡”‡ Šƒ”˜‡•–‡† ˆ‘ŽŽ‘™‡† „› •–ƒ‹‹‰ ‡ŽŽ• ˆ‘” ˆŽ‘™ ›–‘‡–”› ƒ• †‡• ”‹„‡† „‡Ž‘™Ǥ ‡ †ƒ› —Ž–—”‡• ‹ Ž—†‡† ƒ††‹–‹‘ ‘ˆ ”‡ ‘„‹ƒ–

(42) Ǧ͹ ȋͷ ‰ȀŽǡ ǡ‡‡Ž—šȌ‘†ƒ›Ͳƒ†”‡ ‘„‹ƒ–

(43) ǦʹȋͳͲȀŽǡ‡–—•ǡ‡”›˜‹ŽŽ‡ǡǡȌ ‘†ƒ›•‡˜‡ǢŠƒ”˜‡•–‹‰‘ˆ ‡ŽŽ•‘ —””‡†ƒ–†ƒ›ͳͲǤ CD4Tcelllinestimulation:  ‡ŽŽ•ˆ”‘M.tuberculosisƒ–‹‰‡•’‡ ‹ˆ‹ Ǧ ‡ŽŽŽ‹‡•ȋͳǤͷšͳͲͶȀ™‡ŽŽȌ™‡”‡ —Ž–—”‡† ‹ –”‹’Ž‹ ƒ–‡ ‹ —Ž–—”‡ ‡†‹— ™‹–Š ƒ—–‘Ž‘‰‘—• ‘” Ǧ ƒ– Š‡†Ȁ‹•ƒ– Š‡† ƒ•ȋͷšͳͲͶȀ™‡ŽŽǡ‹””ƒ†‹ƒ–‡†ȋʹͲͲͲ”ƒ†Ȍ‹ͻ͸Ǧ™‡ŽŽ•ˆŽƒ–Ǧ„‘––‘‡†‹ ”‘–‹–‡” ’Žƒ–‡•‹–Š‡’”‡•‡ ‡‘”ƒ„•‡ ‡‘ˆƒ–‹‰‡ƒ† —Ž–—”‡†ƒ–͵͹‘ƒ†ͷΨʹǤ–†ƒ› –Š”‡‡ǡ•—’‡”ƒ–ƒ–•™‡”‡ ‘ŽŽ‡ –‡†ˆ‘”–Š‡†‡–‡ –‹‘‘ˆ

(44) JȋʹȌǤ  FlowCytometry ‡ŽŽ• ™‡”‡ •–ƒ‹‡† ™‹–Š ƒ–‹Ǧ͵Ǧ‡”ǡ ƒ–‹ǦͶǦ ƒ† ƒ–‹ǦͺǦ ȋ ‹‘• ‹‡ ‡•ǡ ”‡„‘†‡‰‡ǡ ‡Ž‰‹—Ȍ ˆ‘” ͵Ͳ ‹—–‡• ƒ– ͶιǤ ‡ŽŽ• ™‡”‡ ™ƒ•Š‡† ‹ ȀͲǤͳΨƒ†ˆ‹š‡†‹ͳΨ’ƒ”ƒˆ‘”ƒŽ†‡Š›†‡ȋŠƒ”ƒ ›ǡŠ‡‡–Š‡”Žƒ†•Ȍ ƒ†ƒƒŽ›œ‡†‘ƒ ƒŽ‹„—”ȋ‹‘• ‹‡ ‡•ȌǤƒŽ›•‹•™ƒ•†‘‡—•‹‰‡ŽŽ—‡•– ”‘•‘ˆ–™ƒ”‡ȋ‹‘• ‹‡ ‡•ȌǤ  Analysisofproliferatinglymphocytes(CFSE) ‡ŽŽ•‰ƒ–‡†‘Ž‹˜‡Ž›’Š‘ ›–‡• ‘„‹‡†™‹–Š‰ƒ–‹‰‘͵Ϊ ‡ŽŽ•™‡”‡ƒƒŽ›œ‡† ˆ‘” ’”‘Ž‹ˆ‡”ƒ–‹‘ǤŠ‡'‰‡‘‡–”‹ ‡ƒ™ƒ•—•‡†ƒ•ƒ‡ƒ•—”‡‘ˆ’”‘Ž‹ˆ‡”ƒ–‹‘ ƒ† ƒŽ —Žƒ–‡†ƒ•ˆ‘ŽŽ‘™•ǣ'‰‡‘‡–”‹ ‡ƒα‰‡‘‡–”‹ ‡ƒȋ‘Ǧ’”‘Ž‹ˆ‡”ƒ–‡† ‡ŽŽ• ‡ŽŽ•ȌǦ‰‡‘‡–”‹ ‡ƒȋ–‘–ƒŽ ‡ŽŽ•ȌǤŠ‡'‰‡‘‡–”‹ ‡ƒ™ƒ•–Š‡—•‡†–‘ ƒŽ —Žƒ–‡ –Š‡”‡Žƒ–‹˜‡’”‘Ž‹ˆ‡”ƒ–‹‘™Š‹ Š‹•‹ˆƒ ––Š‡’‡” ‡–ƒ‰‡‘ˆ–Š‡ƒš‹ƒŽ’”‘Ž‹ˆ‡”ƒ–‹‘ ȋ Ȍǡ ‘””‡ –‡† ˆ‘” •’‘–ƒ‡‘—• ’”‘Ž‹ˆ‡”ƒ–‹‘ ȋ

(45) Ǧͳ ’ͳ͹ ƒ‰͹͹ȂͺͷȌǣ ȋȋȟ ‰‡‘‡–”‹  ‡ƒ •ƒ’Ž‡Ǧ ȟ ‰‡‘‡–”‹  ‡ƒ ‘–”‘Ž ’‡’–‹†‡ȌȀȋȟ ‰‡‘‡–”‹  ‡ƒ  Ǧ ȟ ‰‡‘‡–”‹ ‡ƒ ‘–”‘Ž’‡’–‹†‡ȌȌȗͳͲͲΨαΨ‘ˆƒš‹ƒŽ’”‘Ž‹ˆ‡”ƒ–‹‘ǤŠ‡ —–‘ˆˆˆ‘” ƒ ’‘•‹–‹˜‡ ’”‘Ž‹ˆ‡”ƒ–‹‘ ™ƒ• •‡– ƒ– ͳͲΨ ”‡Žƒ–‹˜‡ ’”‘Ž‹ˆ‡”ƒ–‹‘Ǥ Š‹• ƒ”„‹–”ƒ”› Š‹‰Š –Š”‡•Š‘Ž†˜ƒŽ—‡™ƒ• Š‘•‡‹‘”†‡”–‘Ž‹‹––Š‡—„‡”‘ˆ ƒ†‹†ƒ–‡‡’‹–‘’‡•–‘„‡ ‡˜ƒŽ—ƒ–‡†‹•—„•‡“—‡–‡š’‡”‹‡–•Ǥ  IFNJJELISA Š‡ ‘ ‡–”ƒ–‹‘

(46) J ‹ –Š‡ •—’‡”ƒ–ƒ–• ™ƒ• ‡ƒ•—”‡† „› 

(47)  ȋǦ›‡ Šǡ –”‡ Š–ǡŠ‡‡–Š‡”Žƒ†•Ȍƒ ‘”†‹‰–‘–Š‡ƒ—ˆƒ –—”‡”ǯ•‹•–”— –‹‘•ǤŠ‡†‡–‡ –‹‘ Ž‹‹–‘ˆ–Š‡ƒ••ƒ›‹•ʹͲ’‰ȀŽ

(48) ɀǤƒ’Ž‡•™‡”‡–‡•–‡†‹†—’Ž‹ ƒ–‡Ǣ–Š‡‡ƒ˜ƒŽ—‡ ‘ˆ —•–‹—Žƒ–‡† —Ž–—”‡• ™ƒ• •—„–”ƒ –‡† ˆ”‘ –Š‡ ‡ƒ ˜ƒŽ—‡ ‘ˆ –Š‡ •–‹—Žƒ–‡† —Ž–—”‡•Ǥ

(49) J”‡•’‘•‡tͳͲͲ’‰ȀŽ™ƒ• ‘•‹†‡”‡†’‘•‹–‹˜‡ˆ‘”ƒ†ˆ‘”M. tuberculosis ƒ–‹‰‡ •’‡ ‹ˆ‹   ‡ŽŽ Ž‹‡• ™Š‡

(50) J ”‡•’‘•‡ ™ƒ• t ͷͲ ’‰ȀŽ —Ž‡•• ‹†‹ ƒ–‡†‘–Š‡”™‹•‡Ǥ . ͸ʹ.

(51) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. Chromiumreleaseassay •–ƒ”‰‡– ‡ŽŽ•ǡŠ—ƒǦ ȋ ǦȗͲʹͲͳǡǦ͹ǡǦ™͹ȌǡǦ Ǧ˜ͳ͹͵͵ Ǧ. ȋǦ –”ƒ•†— ‡†™‹–Šƒ‡š’”‡••‹‘˜‡ –‘”‡ ‘†‹‰–Š‡M.tuberculosis ‘•‰‡‡˜ͳ͹͵͵ ƒ†–Š‡ ƒ”‡”ȋ͵ͳǡ͵͵ȌȌƒ†Ǧ Ǧ ȋǦ  –”ƒ•†— ‡†™‹–Šƒ‡’–› ‘–”‘Ž˜‡ –‘”‡ ‘†‹‰‘Ž› Ȍ™‡”‡‹ —„ƒ–‡†ƒ–͵͹ι ˆ‘”ͳŠ”™‹–ŠͲǤͳ‹ƒʹͷͳ”Ͷȋ‡”•Šƒǡ‹–‡†‹‰†‘Ȍǡ™ƒ•Š‡†ǡƒ†’Žƒ–‡†‹ –”‹’Ž‹ ƒ–‡‹ͻ͸Ǧ™‡ŽŽ”‘—†Ǧ„‘––‘’Žƒ–‡•ȋʹͷͲͲ ‡ŽŽ•Ȁ™‡ŽŽȌǤˆˆ‡ –‘”ͺΪ ‡ŽŽ•™‡”‡ ƒ††‡† ‹ †‹ˆˆ‡”‡– ‡ˆˆ‡ –‘”Ǧ–‘Ǧ–ƒ”‰‡– ȋǣȌ ”ƒ–‹‘•ǡ ͳʹǣͳǡ ʹͷǣͳ ƒ† ͷͲǣͳ –‘‰‡–Š‡” ™‹–Š ‡‹–Š‡”‡†‹—ǡ’‡’–‹†‡ȋʹͷP‰ȀŽȌǡ‘”ͷΨ”‹–‘ǦͳͲͲƒ†™‹–Š‘ˆ™‹–Š‘—–’ƒ Ǧ Žƒ••

(52) ‘” Ǧ Žƒ••

(53)

(54) ƒ–‹„‘†‹‡•Ǥˆ–‡”•‹šŠ‘—”•–Š‡•—’‡”ƒ–ƒ–•™‡”‡Šƒ”˜‡•–‡†ǡƒ† –Š‡’‡” ‡–ƒ‰‡Ž›•‹•™ƒ• ƒŽ —Žƒ–‡†ƒ•ˆ‘ŽŽ‘™•ǣ ȏȋ”‡Ž‡ƒ•‡Ǧ•’‘–ƒ‡‘—•”‡Ž‡ƒ•‡ȌȀȋƒš‹—”‡Ž‡ƒ•‡Ǧ•’‘–ƒ‡‘—•”‡Ž‡ƒ•‡ȌȐȗͳͲͲΨǤ  . Results SelectionofrecombinantM.tuberculosisproteinsandcorrespondingpeptides ‘” –Š‹• •–—†› ™‡ •‡Ž‡ –‡† ˆ‘—” M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ’”‡˜‹‘—•Ž› •Š‘™ –‘ „‡ ’”‘‹‡–Ž› ”‡ ‘‰‹œ‡† ‹ Š—ƒ•ǡ ‹ ‘”†‡” –‘ ‹†‡–‹ˆ› ’‘–‡–‹ƒŽ ͶΪ ƒ† ͺΪ  ‡ŽŽ ”‡•’‘•‡• ƒ• ™‡ŽŽ ƒ• –‘ ‹†‡–‹ˆ› ’‡’–‹†‡ ‡’‹–‘’‡• ”‡ ‘‰‹œ‡† ‹ –Š‡ ‘–‡š– ‘ˆ •’‡ ‹ˆ‹  Ǧ Žƒ••

(55)  ƒ† Žƒ••

(56)

(57)  ‘Ž‡ —Ž‡•Ǥ Š‡ ‹—‘†‘‹ƒ–ǡ‡ƒ”Ž›’Šƒ•‡•‡ ”‡–‡†ƒ–‹‰‡‰ͺͷ™ƒ•–ƒ‡ƒŽ‘‰ƒ•”‡ˆ‡”‡ ‡ ƒ–‹‰‡Ǥ M. tuberculosis ‘• ƒ–‹‰‡• ˜ͳ͹͵͵ ǡ ˜ʹͲʹͻ  ƒ† ˜ʹ͸ʹ͹  ™‡”‡ Š‘•‡ ‘ –Š‡ „ƒ•‹• ‘ˆ  ‡ŽŽ ”‡ ‘‰‹–‹‘ ’”‘ˆ‹Ž‡• ‹ M. tuberculosis ‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ• ȋ ’ƒ–‹‡–•ƒ†–—„‡” —Ž‹•‹–‡•–ȋȌ ‘˜‡”–‡”•Ȍˆ”‘‘—”’”‡˜‹‘—•™‘”ȋͳͺȌǣƒŽŽ –Š”‡‡ƒ–‹‰‡•”ƒ‡†ƒ‘‰–Š‡–‘’ͳͲ‘•–ˆ”‡“—‡–Ž›”‡ ‘‰‹œ‡†ƒ–‹‰‡•‹–Š‡M. tuberculosis ‹ˆ‡ –‡† ‰”‘—’•Ǥ M. tuberculosis ‘• ƒ–‹‰‡ ˜ʹͲ͵ͳ  ȋ •’ǡ Š•’ͳ͸ǡ ƒǦ ”›•–ƒŽŽ‹Ȍ™ƒ•‹ Ž—†‡†•‹ ‡ƒ›•–—†‹‡•Šƒ˜‡ƒ††”‡••‡†–Š‡”‘Ž‡‘ˆ–Š‹•Š‡ƒ–Ǧ•Š‘  ’”‘–‡‹ ‹ ƒ–‹Ǧ› ‘„ƒ –‡”‹ƒŽ ‹—‹–› ȋͳͷǡ͵ͻǡͶͳȌǤ ‡ ‘„‹ƒ– ’”‘–‡‹• ȋƒ„Ž‡ ͳȌ ƒ† ‘˜‡”Žƒ’’‹‰ •‡–• ‘ˆ ’‡’–‹†‡• ‘ˆ –Š‡•‡ ˆ‹˜‡ ƒ–‹‰‡• ™‡”‡ ’”‡’ƒ”‡† ȋ•‡‡ •—’’Ž‡‡–ƒ”›ˆ‹Ž‡ͳˆ‘”ƒŽŽ’‡’–‹†‡•‡“—‡ ‡•ȌǤ  Table1.SelectedMycobacteriumtuberculosis antigenstestedinpresentstudy. ‘•‰‡‡•. ”‡ˆ‡”‡ ‡‰‡‡ a. ˜—„‡”. ‡‡ ƒ‡a. ˜ͳ͹͵͵ ˜ʹͲʹͻ ˜ʹͲ͵ͳ ˜ʹ͸ʹ͹ . pfkB hspX. ˜ͳͺͺ͸ . fbpB. ‘Ž‡ —Žƒ” ƒ••ȋƒȌa. ”‘†— –a. ‡ˆ‡”‡ ‡•. ʹʹǡͶ ͵ͷǡͶ ͳ͸ǡ͵ Ͷ͸ǡ͵. ‘•‡”˜‡†–”ƒ•‡„”ƒ‡’”‘–‡‹ ’Š‘•’Š‘ˆ”— –‘‹ƒ•‡ˆ Š‡ƒ–•Š‘ ’”‘–‡‹ •’ȋƒŽ’ŠƒǦ ”›•–ƒŽŽ‹Ȍ ‘•‡”˜‡†Š›’‘–Š‡–‹ ƒŽ’”‘–‡‹. ͸ǡͳͲ ͸ǡͳͲǡͳͶ ͸ǡͻǡͳͲǡͳͶǡʹͳǦʹ͵ ͸ǡͳͲǡͳͶ. ͵Ͷǡ͸. •‡ ”‡–‡†ƒ–‹‰‡ͺͷǦˆ„’ ȋ› ‘Ž›Ž–”ƒ•ˆ‡”ƒ•‡ͺͷǡ‰ͺͷȌ. ʹͷ. ‘–ƒ–‹‘•ƒ”‡ˆ”‘™™™Ǥ–„†„Ǥ‘”‰ƒ†Š––’ǣȀȀ‰‡‘Ž‹•–Ǥ’ƒ•–‡—”Ǥˆ”Ȁ—„‡” —‹•–Ȁ.    . ͸͵.

(58) Chapter3.  IdentificationofM.tuberculosisinducedantigenspecificCD4+andCD8+Tcell responsesbasedonCFSEproliferation ‡—•‡† „ƒ•‡†’”‘Ž‹ˆ‡”ƒ–‹‘‘ˆǡ•‹ ‡–Š‹•‡–Š‘†ƒŽŽ‘™•–”ƒ ‹‰‘ˆ„‘–Š ͶΪ ƒ† ͺΪ  ‡ŽŽ •—„•‡– ”‡•’‘•‡• ‹ –Š‡ •ƒ‡ ‡ŽŽ ’‘’—Žƒ–‹‘ ȋʹͲȌǤ ‹‰—”‡ ͳ †‡‘•–”ƒ–‡• –›’‹ ƒŽ   „ƒ•‡† ͶΪ ƒ† ͺΪ  ‡ŽŽ ”‡•’‘•‡ ’”‘ˆ‹Ž‡• –‘ M. tuberculosis ƒ–‹‰‡• ƒ† ‘–”‘Ž ‘†‹–‹‘• ™Š‡ ƒ••ƒ›‹‰  ˆ”‘ ƒ  ”‡•’‘•‹˜‡ †‘‘”Ǥ ‘ŽŽ‘™‹‰ •–‹—Žƒ–‹‘ ‘ˆ ‡ŽŽ• ™‹–Š ǡ •‹‰‹ˆ‹ ƒ– ͶΪ ƒ† –‘ ƒ Ž‡••‡”‡š–‡–ƒŽ•‘ͺΪ ‡ŽŽ”‡•’‘•‡• ‘—Ž†„‡‘„•‡”˜‡†ǡ—†‡”Ž‹‹‰–Š‡•‹‰‹ˆ‹ ƒ– ƒ†˜ƒ–ƒ‰‡ ‘ˆ –Š‹• ƒ••ƒ› ƒŽŽ‘™‹‰ ”‡•’‘†‹‰ •—„’‘’—Žƒ–‹‘• –‘ „‡ ’Š‡‘–›’‡† ƒ† Šƒ”ƒ –‡”‹œ‡†Ǥ ‘ ”‡•’‘•‡ ™ƒ• •‡‡ –‘ –Š‡ ‡‰ƒ–‹˜‡ ‘–”‘Ž ‡†‹— ‘” –‘ –Š‡ ‹””‡Ž‡˜ƒ– ‘–”‘Ž ’‡’–‹†‡

(59) Ǧ‰ƒ‰͹͹Ȃͺͷ Ǥ ‘ŽŽ‘™‹‰ •–‹—Žƒ–‹‘ ‘ˆ  ™‹–Š M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡ ˜ͳ͹͵͵  ‘” ‹–• ‘””‡•’‘†‹‰ ’‡’–‹†‡• ’”‘Ž‹ˆ‡”ƒ–‹˜‡ͶΪ ‡ŽŽ”‡•’‘•‡•™‡”‡‘„•‡”˜‡†ȋ ‹‰—”‡ͳƒȌǤ   „ ƒ 

(60) ‰ƒ‰͹͹Ǧͺͷ

(61) ‰ƒ‰͹͹Ǧͺͷ  0%  0%          74.4%  100% 100% 39.2%   ˜ͳ͹͵͵ ͳͶͳǦͳ͸Ͳ ‰ͺͷͳ͸ͳǦͳͺͲ ‰ͺͷͳͺͳǦʹͲͲ  ˜ͳ͹͵͵  30.4% 26.9% 53.7% 21.7%    ˜ͳ͹͵͵ ͳ͸ͳǦͳͺͲ ˜ʹͲ͵ͳ ͻͳǦͳͳͲ ˜ʹͲ͵ͳ ͳͳͳǦͳ͵Ͳ ˜ͳ͹͵͵ ͺͳǦͳͲͲ  18.1% 0.1% 13.5%  61.1%   CFSE   Figure 1. Proliferative T cell response profiles to M. tuberculosis antigens compared to control conditions.  PBMCfromdonor9(Table2,HLAA2,A24,B35,B57,C4,C6,DR7,DR14)werestimulatedwithPHAorHIVgag 77  85b aspositiveandnegativecontrols,respectively,orwithM.tuberculosistestantigens.After6daysofculturingCFSE  +(a)orCD8+Tcells(b)wasmeasured:CD4+orCD8+TcellsweregatedfromaCD3+ Tcellgate proliferationofCD4  combined with a live lymphocyte gate. Individual histogram plots show the % relative proliferation (( geometric mean sample   geometric mean control peptide)/ ( geometric mean PHA  geometric mean control  peptide))*100% = % of maximal proliferation. PBMC were stimulated with Rv1733c and corresponding peptides,  someofwhichinducedCD4+Tcellproliferationwhileothersdidnot(a)PBMCwerealsostimulatedwithfour20mer  peptides of Ag85B and Rv2031c encoding identified and published HLAA2 restricted epitopes (see text) to which +TcellproliferationwasobservedinthisHLAA2positivedonor(b).Arelativeproliferation t10%was  indeedCD8 40. 30. 30. 20. 20 10. 10. 100. 101. 102 CFSE donor 32.003. 103. 104. 100. 101. donor 32.004. 102 CFSE donor 32.003. 103. 104. 50. 50. 40. 40. 40. 40. 30. 30. 30. 30. 20. 20. 20. 20. 10. 10. 10. 50. 100. counts. donor 32.005. ‘–”‘Ž ‘†‹–‹‘•. 50. 40. 101. 102 CFSE donor 32.006. 103. 100. 104. 50. 50. 40. 40. 30. 30. 20. 20. 10. 10. 100. 101. 102. 103. 40. 30. 30. 20. 20. 10. 10. 101. 102. 103. consideredpositive.. ͸Ͷ. 104. 104. 10. 100. 101. 50. 101. 102. 103. 102 CFSE donor 32.158. 103. 104. 40. 30. 30. 20. 20. 101. 102 CFSE. 101. 50. 103. 104. 101. 102 CFSE donor 32.160. 103. 104. 100. 101. 102 CFSE donor 32.073. 103. 104. 100. 101. 103. 104. 10. 100. 102 CFSE donor 32.071. 103. 104. 50. 40. 40. 30. 30. 20. 20. 10. 100. 100. 50. 40. 104. 50. 40. 100. 103. donor 32.015. donor 32.023. 50. 102 CFSE donor 32.021. 10. 100. 104. 101. donor 32.004. 50. 10. 100. 101. 102 CFSE. 103. 104. 102 CFSE. M.tuberculosisƒ–‹‰‡•. donor 32.005. 50.

(62) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. ‡ ™ƒ–‡† –‘ ‡•–ƒ„Ž‹•Š ™Š‡–Š‡” ‘—” ‡–Š‘† ™ƒ• ƒ„Ž‡ –‘ ‘ˆ‹” ƒŽ”‡ƒ†› ’—„Ž‹•Š‡† ‡’‹–‘’‡•‘ˆ‰ͺͷƒ†˜ʹͲ͵ͳ ȋ •’ǡDǦ ”›•–ƒŽŽ‹Ȍǡ‹‘”†‡”–‘ˆ—”–Š‡”˜ƒŽ‹†ƒ–‡–Š‡   „ƒ•‡† ’”‘Ž‹ˆ‡”ƒ–‹‘ ‡–Š‘†Ǥ ‘” –Š‡ ƒ–—”‡ ‰ͺͷ ’”‘–‡‹ǡ ˆ‹˜‡ ǦȗͲʹͲͳ ‡’‹–‘’‡• Šƒ˜‡ „‡‡ †‡• ”‹„‡† ȋ‰ͺͷ’ͶͷǦͷ͵ ǡ ‰ͺͷ’ͳ͸͸Ǧͳ͹ͷ  

(63) ǡ ‰ͺͷ’ͳͺ͵Ǧͳͻʹ

(64)  ǡ ‰ͺͷ’ͳͻͺǦʹͲ͸  

(65)  ƒ† ‰ͺͷ’ʹ͵ͻǦʹͶ͹  ȋͳ͸ȌȌǤ ‡ –Š‡”‡ˆ‘”‡ ‹ Ž—†‡† ʹͲǦ‡” ’‡’–‹†‡• ‘–ƒ‹‹‰ –Š‡•‡ ˆ‹˜‡ ǦȗͲʹͲͳ ”‡•–”‹ –‡†‡’‹–‘’‡•‹‘—”•–—†›Ǥ ‘—”‘—–‘ˆ–Š‡ˆ‹˜‡ʹͲǦ‡”•™‡”‡‹†‡‡†”‡ ‘‰‹œ‡† „›ͺΪ ‡ŽŽ•ˆ”‘–Š‹•†‘‘”ȋ ‹‰—”‡ͳ„Ȍƒ•™‡ŽŽƒ•„›‘–Š‡” ǦʹΪ‹†‹˜‹†—ƒŽ• ȋ†ƒ–ƒ‘–•Š‘™ȌǤ Ž•‘ ˆ‘” ˜ʹͲ͵ͳ  –Š”‡‡ ǦȗͲʹͲͳ ”‡•–”‹ –‡† ‡’‹–‘’‡• Šƒ˜‡ ’”‡˜‹‘—•Ž› „‡‡ ‹†‡–‹ˆ‹‡†ǣ˜ʹͲ͵ͳ ’ʹͳǦʹͻ   ȋ͸Ȍǡ˜ʹͲ͵ͳ ’ͻͳǦͳͲͷ   ȋͳͷȌƒ† ˜ʹͲ͵ͳ ’ͳʹͲǦͳʹͺ

(66)  ȋͶǡ͸ȌǤ —” †ƒ–ƒ •Š‘™ –Šƒ– ʹͲǦ‡”• ‡ ‘†‹‰ ‡’‹–‘’‡• ˜ʹͲ͵ͳ ’ͻͳǦͳͲͷ ƒ† ˜ʹͲ͵ͳ ’ͳʹͲǦͳʹͺ ȋ„—– ‘– ˜ʹͲ͵ͳ ’ʹͳǦʹͻ ‹ –Š‹• †‘‘”Ȍ ™‡”‡ „‘–Š ”‡ ‘‰‹œ‡†„›•‡˜‡”ƒŽ ǦʹΪ‹†‹˜‹†—ƒŽ•Ǥ ‹‰—”‡ͳ„†‡‘•–”ƒ–‡•’”‘Ž‹ˆ‡”ƒ–‹˜‡ͺΪ  ‡ŽŽ”‡•’‘•‡•ˆ‘ŽŽ‘™‹‰•–‹—Žƒ–‹‘™‹–Š•‡˜‡”ƒŽ‘ˆ–Š‡ƒ„‘˜‡‡–‹‘‡†ʹͲǦ‡”M. tuberculosis’‡’–‹†‡•Ǥ Ž•‘ ’‡’–‹†‡• ‡ ‘†‹‰ ’”‡˜‹‘—•Ž› ’—„Ž‹•Š‡†ǡ  Žƒ••

(67)

(68)  ’”‡•‡–‡† ‡’‹–‘’‡• ™‡”‡ ”‡ ‘‰‹œ‡† ‹ –Š‹• ƒ••ƒ›Ǥ Š‡•‡ ‹ Ž—†‡†ǣ ˜ʹͲ͵ͳ  ’‡’–‹†‡ Ͷ ‡ ‘†‹‰ ˜ʹͲ͵ͳ ’͵ͳǦͷͲ ȋ  ǡ ǦͳȗͲ͵ͲͳȌȋͻǡͳͷǡʹͳȌƒ†˜ʹͲ͵ͳ ’‡’–‹†‡•͵ǡͻǡ ƒ† ͳͲ ™Š‹ Š ‡ ‘†‡ •‡˜‡”ƒŽ ‡’‹–‘’‡• ”‡ ‘‰‹œ‡† ‹–Š‡ ‘–‡š– ‘ˆ ǦͳȗͳͷͲͳ ȋͳǡͷȌ ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ ƒ‡ –‘‰‡–Š‡”ǡ –Š‡ ”‡•—Ž–• ˆ”‘ –Š‡   ƒ••ƒ› ƒ‰”‡‡ ™‡ŽŽ ™‹–Š ”‡•—Ž–• ˆ”‘ ‘–Š‡” ƒ••ƒ›•ǡ ‹ Ž—†‹‰ –Š‡ ƒ„‹Ž‹–› –‘ ‹†‡–‹ˆ› ’‡’–‹†‡ ‡’‹–‘’‡• ”‡ ‘‰‹œ‡†„›ͶΪƒ†ͺΪ ‡ŽŽ•Ǥ  ImmunogenicityofM.tuberculosisDosRregulonencodedantigensbasedon CFSEproliferationandflowcytometry   Žƒ„‡ŽŽ‡†  ˆ”‘ ”ƒ†‘Ž› •‡Ž‡ –‡†ǡ Š‡ƒŽ–Š›ǡ Ǧ”‡•’‘•‹˜‡ „Ž‘‘† „ƒ †‘‘”•™‡”‡•–‹—Žƒ–‡†™‹–ŠM.tuberculosisŽ›•ƒ–‡ǢǢM.tuberculosis‘•”‡‰—Ž‘ ‡ ‘†‡† ’”‘–‡‹• ˜ͳ͹͵͵ ǡ ˜ʹͲʹͻ ǡ ˜ʹ͸ʹ͹ ǡ ˜ʹͲ͵ͳ Ǣ ‰ͺͷ ’”‘–‡‹Ǣ ƒ† ƒŽŽ ”‡•’‡ –‹˜‡•‹‰Ž‡’‡’–‹†‡•ˆ”‘‡ƒ Š‘ˆ–Š‡ͷ”‡ ‘„‹ƒ–ƒ–‹‰‡•Ǥƒ„Ž‡ʹ•Š‘™•–Š‡  Žƒ••ǦǡȂǡǦƒ†Ǧ‰‡‘–›’‡•ƒ†–Š‡ ‘””‡•’‘†‹‰invitro”‡ƒ –‹˜‹–›‘ˆ –Š‡†‘‘”•Ǥ

(69) J”‡•’‘•‡•–‘‡š ‡‡†‹‰ͳͲͲ’‰ȀŽ™‡”‡ ‘•‹†‡”‡†’‘•‹–‹˜‡ǡƒ• —•‡†‹‘—”’”‡˜‹‘—••–—†‹‡•ȋʹǡͳͺǡͳͻȌǤ

(70) —‘‰‡‹ ”‡‰‹‘• ‘—Ž†„‡‹†‡–‹ˆ‹‡†™‹–Š‹–Š‡•‡“—‡ ‡•‘ˆ–Š‡•‡M.tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ȋƒ„Ž‡ ͵ƒǦ ȌǤ ƒ„Ž‡ ͵ƒ ƒ† ͵„ •Š‘™ –Š‡ ‘’”‡Š‡•‹˜‡ ”‡ ‘‰‹–‹‘ ’”‘ˆ‹Ž‡• ‘ˆ ͶΪ ƒ† ͺΪ  ‡ŽŽ• ‘ˆ –Š‡ –‡•–‡† ƒ–‹‰‡• ƒ† –Š‡‹” ”‡•’‡ –‹˜‡ •‹‰Ž‡ ’‡’–‹†‡• ‹ ƒŽŽ †‘‘”• •–—†‹‡† Š‡”‡Ǥ • ƒ–‹ ‹’ƒ–‡†ǡ ”‡•’‘•‡• –‘ –Š‡ ™Š‘Ž‡ ”‡ ‘„‹ƒ– ’”‘–‡‹• ™‡”‡ ‘•–Ž› ‘ˆ‹‡† –‘ –Š‡ ͶΪ  ‡ŽŽ•Ǥ

(71)  •‡˜‡”ƒŽ ƒ•‡• ͶΪ  ‡ŽŽ ”‡•’‘•‡• ™‡”‡ ƒ ‘’ƒ‹‡† „› ͺΪ  ‡ŽŽ ”‡•’‘•‡• ȋ‡Ǥ‰Ǥ ‹ †‘‘” ͳȌǡ ™Š‹Ž‡ ‹•‘Žƒ–‡† ͺΪ  ‡ŽŽ”‡•’‘•‡• –‘–Š‡ ”‡ ‘„‹ƒ– ’”‘–‡‹•™‡”‡‘–‘„•‡”˜‡†Ǥ •‡š’‡ –‡†ǡ–Š‡”‡™‡”‡•—„•–ƒ–‹ƒŽ‹–‡”Ǧ‹†‹˜‹†—ƒŽ†‹ˆˆ‡”‡ ‡•‹ƒ–‹‰‡ƒ†’‡’–‹†‡ ”‡ ‘‰‹–‹‘ǡ •‹ ‡ •‘‡ †‘‘”• ”‡•’‘†‡† –‘ ƒ› M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡†’”‘–‡‹•ƒ† ‘””‡•’‘†‹‰’‡’–‹†‡•™Š‡”‡ƒ•‘–Š‡”•‘Ž›”‡•’‘†‡†–‘ˆ‡™ ’”‘–‡‹•‘”’‡’–‹†‡•Ǥ‡˜‡”–Š‡Ž‡••ǡƒŽŽͳͷΪ†‘‘”•”‡•’‘†‡†–‘ƒ–Ž‡ƒ•–‘‡‘ˆ–Š‡ ”‡ ‘„‹ƒ–’”‘–‡‹•ƒ†Ȁ‘”’‡’–‹†‡•‹˜‡•–‹‰ƒ–‡†Ǥ. ͸ͷ.

(72) Chapter3 Table2.HLAclassA,B,Cand–DRgenotypesandIFNresponsesto M.tuberculosis PPDofdonorsstudiedincurrentstudy HLAtype. Donor number ͳ ʹ ͵ Ͷ ͷ ͸ ͹ ͺ ͻ ͳͲ ͳͳ ͳʹ ͳ͵ ͳͶ ͳͷ. HLAA ͳ ʹǡ͵ͳ ͳͳǡ͵Ͳ ʹǡʹͻ ʹ ʹǡ͵ ʹ ͵ǡ͵ͳ ʹǡʹͶ ͵ǡʹͶ ͳǡ͵ͳ ʹǡ͸ͺ ͵ ͵ǡʹͶ ͵ǡͳͳ. HLAB ͹ǡͺ ͺǡͶͶ ͳͺǡʹ͹ ͶͷǡͷͲ ͶͶǡ͵ͻ ͷͳǡ͹ ͹ǡͺ ͷ͸ǡ͵ͷ ͵ͷǡͷ͹ ͳͶǡͳͺ ͷͳǡͷʹ ͶͶǡͷͳ ͹ǡͷͳ ͹ǡ͸ʹ ͳͷǡ͵ͺ. HLAC ͹ ͷǡ͹ ͳǡͷ ͵ǡ͸ ͷǡ͹ ȗ ͹ ͳǡͶ Ͷǡ͸ ȗ ȗ ͳǡͷ ͹ǡͶ ͹ǡͻ ͳǡͳʹ. HLADR ʹǡ͵ ͵ǡͶ ͵ǡͶ Ͷǡ͹ ͹ǡͳͷ ͳͷǡͳͳ ʹǡ͵ ͳǡͶ ͹ǡͳͶ ͳǡͶ ͳͳǡͳͷ ͳͳǡͳͶ ͳǡͳͷ ͳͳǡͳ͵ ͵ǡͳ͵. PPDa pg/ml Ϊ Ϊ Ϊ Ϊ ΪΪΪ Ϊ Ϊ ΪΪ ΪΪ ΪΪΪ Ϊ Ϊ ΪΪ Ϊ Ϊ. ȗǡ Ǧ–›’‡—‘™Ǥ a, ‡•’‘•‡•–‘ǣΪǡͳͲͲǦͷͲͲ’‰ȀŽǢΪΪǡͷͲͲǦͳͲͲͲ’‰ȀŽǢΪΪΪǡεͳͲͲͲ’‰ȀŽǤ. 

(73)  –Š‹• •‡”‹‡• ‘ˆ †‘‘”•ǡ ”‡ ‘„‹ƒ– ƒ–‹‰‡• ˜ͳ͹͵͵ ǡ ˜ʹͲʹͻ  ƒ† ˜ͳͺͺ͸  ȋ‰ͺͷȌ ™‡”‡ ”‡ ‘‰‹œ‡† ‘•– ‡ˆˆ‹ ‹‡–Ž›ǣ ͹Ȁͳͷ Ϊ †‘‘”• ”‡ ‘‰‹œ‡† ˜ʹͲʹͻ ǡ ͷȀͳͷ ”‡ ‘‰‹œ‡† ˜ͳ͹͵͵  ƒ† ͶȀͳͷ ”‡ ‘‰‹œ‡† ‰ͺͷǤ Š‹• ‘–”ƒ•–‡† –‘ ˜ʹ͸ʹ͹ ǡ ™Š‹ Š™ƒ•”‡ ‘‰‹œ‡†„›‘Ž›ƒ•‹‰Ž‡†‘‘”Ǥ

(74) Ž‹‡™‹–Š‘—”’”‡˜‹‘—•‘„•‡”˜ƒ–‹‘•ǡ ˜ʹͲ͵ͳ ȀŠ•’Ȁacr™ƒ•”‡ ‘‰‹œ‡†„›ƒ‹‘”‹–›‘ˆ–Š‡†‘‘”•ȋͳͺȌǤ ‡’–‹†‡•‹•‡˜‡”ƒŽ‹•–ƒ ‡•ƒ’’‡ƒ”‡†–‘„‡‘”‡ƒ„Ž‡–‘‹†— ‡ͶΪ‘”ͺΪ ‡ŽŽ ”‡•’‘•‡• ™Š‡ ‘’ƒ”‡† –‘ ”‡ ‘„‹ƒ– ’”‘–‡‹•Ǣ –Š‹• ‹‰Š– „‡ †—‡ –‘ –Š‡‹” ’”‡’”‘ ‡••‡†ƒ–—”‡ǡˆƒ ‹Ž‹–ƒ–‹‰Š‹‰Š‡ˆˆ‹ ‹‡ ›ƒ–‹‰‡’”‡•‡–ƒ–‹‘ǡƒ†Ȁ‘”–‘–Š‡‹” Š‹‰Š‡” ‘Žƒ”‹–›ǡ •‹ ‡ ’”‘–‡‹• ƒ† ’‡’–‹†‡• ƒŽŽ ™‡”‡ –‡•–‡† ƒ– ͳͲ ‹ ”‘‰”ƒȀŽ ‘ ‡–”ƒ–‹‘•ǡ”‡‰ƒ”†Ž‡••‘ˆ‘Ž‡ —Žƒ”ƒ••Ǥ

(75) ’‘”–ƒ–Ž›ǡ ƒ •—„•–ƒ–‹ƒŽ —„‡” ‘ˆ ‹—‘‰‡‹  ’‡’–‹†‡• ”‡ ‘‰‹œ‡† „› ͶΪ  ‡ŽŽ•ǡͺΪ ‡ŽŽ•‘”„‘–Š ‘—Ž†„‡‹†‡–‹ˆ‹‡†—•‹‰–Š‹•ƒ’’”‘ƒ ŠǤ‘‡’‡’–‹†‡•™‡”‡ ”‡ ‘‰‹œ‡†„›ͶΪ ‡ŽŽ•ˆ”‘ƒ–Ž‡ƒ•–‘‡–Š‹”†‘ˆ–Š‡†‘‘”•ǡ‡Ǥ‰Ǥ‰ͺͷ’‡’–‹†‡•ͻǡ ͳ͵ȋ͸†‘‘”•Ȍǡ‰ͺͷ’‡’–‹†‡•ͷǡ͸ƒ†˜ʹ͸ʹ͹ ’‡’–‹†‡ͳͻȋͷ†‘‘”•ȌǤ‡˜‡”ƒŽ‘–Š‡” ’‡’–‹†‡•™‡”‡”‡ ‘‰‹œ‡†„›ͶΪ ‡ŽŽ•ˆ”‘ͶȀͳͷ†‘‘”•ǡ•— Šƒ•˜ͳ͹͵͵ ’‡’–‹†‡ ʹǡ˜ʹ͸ʹ͹ ’‡’–‹†‡•ͳͲǡͳͻǡ͵ʹǡ͵Ͷƒ†͵ͷǡƒ†‰ͺͷ’‡’–‹†‡•ͳͲǡͳʹǡͳ͸ƒ†ʹʹǤ Žƒ”‰‡—„‡”‘ˆ’‡’–‹†‡•™ƒ•”‡ ‘‰‹œ‡†„›ƒ–Ž‡ƒ•––Š”‡‡†‘‘”•Ǥƒ”‹‘—•ͺΪ ‡ŽŽ ”‡•’‘•‡•™‡”‡†‡–‡ –‡†ƒ•™‡ŽŽǡƒŽ–Š‘—‰Š–Š‡—„‡”•‘ˆ’‡’–‹†‡•”‡ ‘‰‹œ‡†‘˜‡”ƒŽŽ ™ƒ•Ž‘™‡”–Šƒ–Šƒ–ˆ‘”ͶΪ ‡ŽŽ•ǤŠ‹•‹‰Š–„‡”‡Žƒ–‡†’ƒ”–Ž›–‘–Š‡Ž‡‰–Š‘ˆ–Š‡ ’‡’–‹†‡•ǡ •‹ ‡  Žƒ••

(76)  ƒŽŽ‡Ž‡• –›’‹ ƒŽŽ› ’”‡ˆ‡” ͻǦͳͳǦ‡” ’‡’–‹†‡• ˆ‘” „‹†‹‰ǡ ™Š‡”‡ƒ• –Š‘•‡ ƒ••ƒ›‡† Š‡”‡ ™‡”‡ ʹͲǦ‡”•Ǥ ‡˜‡”–Š‡Ž‡••ǡ ’ƒ”–‹ —Žƒ”Ž› ˆ‘” ˜ͳ͹͵͵  ƒ†‰ͺͷǡ–Š‡”‡™‡”‡•‡˜‡”ƒŽ’‡’–‹†‡•”‡ ‘‰‹œ‡†„›ͺΪ ‡ŽŽ•ˆ”‘ͶǦͷ†‘‘”• Ž‹‡˜ͳ͹͵͵ ’‡’–‹†‡•ͳ͹ȋͷ†‘‘”•Ȍǡʹƒ†ͳͻȋ„‘–ŠͶ†‘‘”•Ȍƒ†‰ͺͷ’‡’–‹†‡•ͷ ƒ†ͳ͵ȋ„‘–ŠͶ†‘‘”•ȌǤ—Ž–‹’Ž‡’‡’–‹†‡•™‡”‡”‡ ‘‰‹œ‡†„›ƒ–Ž‡ƒ•–͵†‘‘”•ǡ•–‹ŽŽ ”‡’”‡•‡–‹‰ʹͲΨ‘ˆ–Š‡’ƒ‡Ž–‡•–‡†Ǥ. ͸͸.

(77) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. Table3a.ProliferativeCD4+TcellresponsetoM.tuberculosis peptidesamongalldonors(n=15,numbers correspondingwithdonorsinTable2).Arelativeproliferation10%wasconsideredpositive.                                  rel. proliferation < 3% rel. proliferation rel. proliferation not tested tested rel. proliferation < 3% rel. proliferation 3-10% 3-10% rel. proliferation >10% >10%  not  Mtb, M. tuberculosis  ƒ„Ž‡͵ •Š‘™•–Š‡ ——Žƒ–‹˜‡”‡ ‘‰‹–‹‘”‡•—Ž–•ˆ‘”„‘–ŠͶΪƒ†ͺΪ ‡ŽŽ•–‘ƒŽŽ –‡•–‡† ƒ–‹‰‡• ƒ† ’‡’–‹†‡• ˆ‘” ƒŽŽ †‘‘”• –‡•–‡†Ǥ Š‡ ”‡•—Ž–• •Š‘™ –Šƒ– ƒ› ’”‘Ž‹ˆ‡”ƒ–‹˜‡ ”‡•’‘•‡•–‘–Š‡ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ƒ„‡ ‹†‡–‹ˆ‹‡†Ǥ Š‹• ƒŽ•‘ —†‡”Ž‹‡• –Š‡ ‹—‘‰‡‹ ‹–› ‘ˆ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡•Ǥ

(78) –‡”‡•–‹‰Ž›ǡ •‡˜‡”ƒŽ ’‡’–‹†‡• ™‡”‡ ”‡ ‘‰‹œ‡† „› „‘–Š ͶΪ ƒ† ͺΪ ‡ŽŽ•ȋ˜ͳ͹͵͵ ǣ’‡’–‹†‡ʹǢ‰ͺͷ’‡’–‹†‡•ͷƒ†ͳ͵ȌǤŠ‹•‹••—‡‹• ‘‘Ž› ‘–ƒ††”‡••‡†‹‡’‹–‘’‡ƒ’’‹‰•–—†‹‡•ǡ•‹ ‡”‡ƒ†Ǧ‘—–•ƒ”‡‘•–Ž›„‹ƒ•‡†–‘™ƒ”†• †‡–‡ –‹‰ͶΪ ‡ŽŽ”‡•’‘•‡•Ǥ —”–Š‡”‘”‡ǡ–Š‡”‡•—Ž–••—‰‰‡•–•‘‡‡”‹ Š‡–‘ˆ ͸͹.

(79) Chapter3 Table3b.ProliferativeCD8+TcellresponsetoM.tuberculosis peptidesamongalldonors(n=15,numbers.  correspondingwithdonorsinTable2).Arelativeproliferation10%wasconsideredpositive.                                   not not tested rel. proliferation < 3% rel. proliferation 3-10% 3-10% rel. proliferation >10% >10% tested rel. proliferation < 3% rel. proliferation rel. proliferation  Mtb, M. tuberculosis  ‡’‹–‘’‡•‹’ƒ”–‹ —Žƒ”Ž›‹—‘‰‡‹ ”‡‰‹‘•ǡˆ‘”‡šƒ’Ž‡˜ͳ͹͵͵ ’‡’–‹†‡•ͳǦ͵ǡͳ͹Ǧ ͳͻǡƒ†‰ͺͷ’‡’–‹†‡•ͷǦͳ͵Ǥ ƒ‡–‘‰‡–Š‡”ǡ™‡ƒ’’Ž‹‡†–Š‡ ‡ŽŽ–”ƒ ‹‰†›‡ –‘˜‹•—ƒŽ‹œ‡ƒ†•‹—Ž–ƒ‡‘—•Ž› “—ƒ–‹ˆ›„‘–ŠͶΪƒ†ͺΪ ‡ŽŽ”‡•’‘•‡•–‘•’‡ ‹ˆ‹ M.tuberculosisƒ–‹‰‡•ƒ† ‹†‹˜‹†—ƒŽ ’‡’–‹†‡•Ǥ ‡ ‹†‡–‹ˆ› ƒ Žƒ”‰‡ •‡”‹‡• ‘ˆ ‡™ ’‘••‹„Ž‡ ’‡’–‹†‡ ‡’‹–‘’‡• Ž‘ ƒ–‡†‘M.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡†ƒ–‹‰‡•ǡ™Š‹ Šƒ”‡”‡ ‘‰‹œ‡†™‹–Š Š‹‰Š‡ˆˆ‹ ‹‡ ›„›ͶΪƒ†Ȁ‘”ͺΪ ‡ŽŽ•Ǥ . ͸ͺ.

(80) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens Table3c.CumulativeCD4 andCD8 TcellresponseprofilestoM.tuberculosis proteinsandtheirrespective  peptidesamongPPDresponsivedonors(n=15). +. +.        Mtb, M. tuberculosis  + Table3d.CumulativeCD4 andCD8+TcellresponseprofilestoM.tuberculosis proteinsandtheirrespective  peptidesamongheterozygousHLADR3+(n=5,CD4+Tcellresponses)andHLAA2+(n=7,CD8+Tcellresponses) PPDresponsivedonors.         M. tuberculosis Mtb,  IdentificationofM.tuberculosisDosRregulonencodedpeptideepitopesinthe contextofgeneticHLAA2andHLADR3polymorphism Š‡ ƒ„‘˜‡ †‡• ”‹„‡† M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ’‡’–‹†‡ ‡’‹–‘’‡• ™‡”‡ ”‡ ‘‰‹œ‡†„›†‹ˆˆ‡”‡–†‘‘”•™‹–Š˜ƒ”›‹‰ –›’‡•Ǥƒ›‘ˆ–Š‡invitro”‡•’‘•‡• ƒ– Š‡†™‹–Šinsilico‡’‹–‘’‡‘–‹ˆ•‡ƒ” Š‡•ˆ‘”–Š‡”‡Ž‡˜ƒ– –›’‡•ȋʹ͸Ȍȋ†ƒ–ƒ‘– •Š‘™ȌǤ Š‹• •—‰‰‡•–• –Šƒ– ”‡•’‘•‡• –‘ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ƒ ƒ–—”ƒŽŽ› ‘ —” ‹ ƒ ™‹†‡ ”ƒ‰‡ ‘ˆ  –›’‡•Ǥ 

(81)  ‘”†‡” –‘ „‡––‡” —†‡”•–ƒ†–Š‡‘Ž‡ —Žƒ”‡ Šƒ‹••‘ˆ‘•”‡‰—Ž‘‡ ‘†‡†‡’‹–‘’‡’”‡•‡–ƒ–‹‘ǡ ™‡ ‡šƒ‹‡† ’‡’–‹†‡ ”‡ ‘‰‹–‹‘ ‹ –Š‡ ‘–‡š– ‘ˆ  Žƒ••

(82)  ƒ† Žƒ••

(83)

(84)  ’‘Ž›‘”’Š‹•Ǥ ‹˜‡ –Š‡ ”‡Žƒ–‹˜‡Ž› Ž‹‹–‡† —„‡” ‘ˆ ‹†‹˜‹†—ƒŽ• ‹ Ž—†‡† ‹ –Š‡ ’ƒ‡Žǡ ™‡ †‡ ‹†‡† –‘ ˆ‘ —• ‘ ʹ Š‹‰ŠŽ› ˆ”‡“—‡– ƒŽŽ‡Ž‡•ǡ ǦȗͲʹͲͳ ƒ† Ǧ ͳȗͲ͵Ͳͳ ȋ͵Ȁͳ͹Ȍ ȋˆ”‡“—‡ ‹‡• ƒ”‡ ̱ͷͲΨ ƒ† ̱ʹͲΨǡ ”‡•’‡ –‹˜‡Ž› ƒ‘‰ ƒ— ƒ•‹ƒ•Ȍȋʹ͵ȌǤ‘‰–Š‡’ƒ‡Ž‘ˆͳͷΪ„Ž‘‘†„ƒ†‘‘”•ǡͷ Ǧ͵’‘•‹–‹˜‡ ƒ†͹ ǦʹΪ†‘‘”•™‡”‡‹†‡–‹ˆ‹‡†Ǥ ‡ŽŽ”‡•’‘•‡•ȋͶΪȌ–‘M.tuberculosis‘• ”‡‰—Ž‘‡ ‘†‡†’‡’–‹†‡•ƒ‘‰–Š‡ͷ Ǧ͵Ϊ†‘‘”•™‡”‡ˆ‘—†‹ʹȀͷ†‘‘”•ǡ ‹ Ž—†‹‰–‘˜ͳ͹͵͵ ’‡’–‹†‡•ͺƒ†ʹǡ˜ʹͲʹͻ ’‡’–‹†‡ʹͲƒ†˜ʹ͸ʹ͹ ’‡’–‹†‡ͳͻ ȋƒ„Ž‡ʹƒ†͵ƒȌǤƒ›’‡’–‹†‡•™‡”‡ƒŽ•‘”‡ ‘‰‹œ‡†„›ȋͺΪ  ‡ŽŽ•ˆ”‘Ȍ ǦʹΪ †‘‘”•Ǥ ‘‡ ’‡’–‹†‡• ™‡”‡ ”‡ ‘‰‹œ‡† „› —’ –‘ ͶȀ͹ ǦʹΪ †‘‘”• •— Š ƒ• ˜ʹ͸ʹ͹  ’‡’–‹†‡• ͳͻ ƒ† ͵ͶǤ  —„‡” ‘ˆ ‘–Š‡” ’‡’–‹†‡• ‹†— ‡† ”‡•’‘•‡• ‹ ͵Ȁ͹ ǦʹΪ †‘‘”•ǣ ˜ʹͲʹͻ  ’‡’–‹†‡ ͵ʹǡ ˜ʹ͸ʹ͹  ’‡’–‹†‡• ͳͲǡ ʹ͸ǡ ͵ʹǡ ͵ͺǦͶͲ ƒ† ˜ʹͲ͵ͳ  ’‡’–‹†‡• Ͷǡ ͹ǡ ͳͲ ƒ† ͳʹ ȋƒ„Ž‡ ʹ ƒ† ƒ„Ž‡ ͵„ȌǤ

(85) –‡”‡•–‹‰Ž›ǡ ˜ʹ͸ʹ͹  ’”‘–‡‹ ™ƒ• ”‡ ‘‰‹œ‡† „› ͶΪ  ‡ŽŽ• ‘ˆ ‘‡ †‘‘” ™Š‡”‡ƒ• –Š‡ •‹‰Ž‡ ’‡’–‹†‡• ‘ˆ ˜ʹ͸ʹ͹  ™‡”‡ ”‡ ‘‰‹œ‡† „› ƒ› ǦʹΪ ƒ† Ǧ͵Ϊ †‘‘”• ‹’Ž›‹‰ –Šƒ– —•‹‰ ’‡’–‹†‡•ƒ›„‡‘”‡•‡•‹–‹˜‡‹ƒ–‹‰‡†‹• ‘˜‡”›–Šƒ—•‹‰‹–ƒ –™Š‘Ž‡ƒ–‹‰‡•Ǥ  . ͸ͻ.

(86) Chapter3 Table4a.ListofM.tuberculosis DosRregulonencodedcandidateHLADR3epitopes epitopesequence. startaa. endaa. scoreinsilico a prediction. Rv1733c.  

(87)  

(88)  

(89) 

(90)  . ͳͷ ͳͳ ͳͶͳ. ʹͻ ʹͷ ͳͷ͸. ʹ͸ ͳͻ ͳͻ. Rv2029c.  .

(91) 

(92)  .          

(93)    . ͵ͷ ͷͳ ͳͳͶ ͳͻͶ ʹ͵ͳ ʹͷʹ. Ͷͻ ͸ͷ ͳʹͺ ʹͲͺ ʹͶͷ ʹ͸͸. ͳͳ ͳʹ ͳͷ ͳͺ ͳͶ ͳ͵. Rv2031c.     

(94) . Ͷ ͳʹͷ. ͳͺ ͳ͵ͻ. ʹ͸ ͳʹ. Rv2627c.         

(95) 

(96) . ͻ͵ ͳͺ͸ ͵ͳ͸. ͳͲͺ ʹͲͲ ͵͵Ͳ. ͳ͹ ͳ͵ ͳ͵. M.tuberculosis DosRantigen. a. a. ǡ’‹–‘’‡‘–‹ˆ• ‘”‡•ƒ† ƒ†‹†ƒ–‡‡’‹–‘’‡•‡“—‡ ‡•ƒ”‡‘„–ƒ‹‡†ˆ”‘™™™Ǥ•›ˆ’‡‹–Š‹Ǥ†‡.   Table4b.ListofM.tuberculosis DosRregulonencodedcandidateHLAA2epitopes M.tuberculosis DosRantigen. epitopesequence. a. startaa. endaa. scoreinsilico a prediction. K D <IC50> mM. b. ELISA c response. CFSE d proliferation. ˜ͳ͹͵͵ .      .   

(97)  

(98) 

(99) 

(100)  

(101) 

(102) . ͺͳ ͹͵ ͹ͺ ͳ͹Ͳ ͳ͸͹ ͳ͸ͳ ͳͺͳ ͳͺͷ ͳͺͺ. ͺͻ ͺͳ ͺ͸ ͳ͹ͺ ͳ͹ͷ ͳ͸ͻ ͳͺͻ ͳͻ͵ ͳͻ͸. ͳͺ ͳͳ ͳͲ ʹͻ ʹͶ ʹ͵ ʹ͸ ʹͲ ʹͲ. εʹͷͲ ͷǡͷ εʹͷͲ Ͳǡͳͳ ͵ ʹǤͺǦ͵Ͷ ͲǡͶʹ ͷ͵ ʹǡͺ. Ǧ Ϊ Ϊ Ϊ Ǧ Ϊ Ϊ Ϊ Ϊ. Ǧ Ǧ Ǧ Ǧ Ǧ Ϊ Ϊ Ǧ Ϊ. ˜ʹͲʹͻ .   . ͵ͳͶ ͵ʹͲ. ͵ʹʹ ͵ʹͺ. ʹͳ ͳʹ. ͳǡͺ εʹͷͲ. Ϊ Ǧ. Ǧ Ǧ. ˜ʹͲ͵ͳ .   .    . ͻͶ ͻ͸ ͳͲͲ. ͳͲʹ ͳͲͶ ͳͲͺ. ͳͻ ͳͺ ͳʹ. ͳ͵ ͷͻ ʹǡͷ. Ǧ Ϊ Ϊ. Ǧ Ǧ Ǧ. ˜ʹ͸ʹ͹ . . . 

(103)  

(104)     

(105) 

(106)     

(107)   . . ͳͷͻ ͳͷͳ ʹͺ͸ ʹͻͳ ʹͺʹ ͵ʹͶ ͵͵ʹ ͵͵͵ ͵͵ͻ ͵ͻͶ ͶͲͲ. ͳ͸͹ ͳͷͻ ʹͻͶ ʹͻͻ ʹͻͲ ͵͵ʹ ͵ͶͲ ͵Ͷͳ ͵Ͷ͹ ͶͲʹ ͶͲͺ. ͳ͹ ͻ ʹͻ ʹͻ ʹͷ ͳͻ ͳͷ ͳͻ ͳͳ ʹͲ ͳ͸. ͶǡͶ ʹͲͻ ͲǡͲͲͶͳ Ͳǡ͹ʹ ͲǡʹͶ ʹͲͲ ͻ Ͷͻ ͺǡ͸ ͵ ʹ͸. Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ϊ Ϊ Ϊ. Ǧ Ǧ Ϊ Ǧ Ǧ Ǧ Ǧ Ϊ Ǧ Ǧ Ǧ. ǡ’‹–‘’‡‘–‹ˆ• ‘”‡•ƒ† ƒ†‹†ƒ–‡‡’‹–‘’‡•‡“—‡ ‡•ƒ”‡‘„–ƒ‹‡†ˆ”‘™™™Ǥ•›ˆ’‡‹–Š‹Ǥ†‡Ǥ ǡ ‘–”‘Ž’‡’–‹†‡ȋ ‘Ž†’‡’–‹†‡Ȍ‹„‹†‹‰ƒ••ƒ›Šƒ†ƒD ‘ˆͲǤͲͲ͸ Ϊ ǡ’‘•‹–‹˜‡

(108) J”‡•’‘•‡–‘ ƒ†‹†ƒ–‡‡’‹–‘’‡ǣΪǡ’‘•‹–‹˜‡”‡•’‘•‡•‡š Ž—•‹˜‡Ž›ˆ‘—†‹ Ǧʹ ȋŠ‡–‡”‘œ›‰‘—•Ȍ†‘‘”•ȋαͳͳȌǢ Ǧǡ’‘•‹–‹˜‡”‡•’‘•‡•ˆ‘—†‹†‘‘”•™‹–Š†‹ˆˆ‡”‡–  Žƒ••ƒŽŽ‡Ž‡•ȋαͷȌǤ Ϊ Ϊ d ǡͺ  ‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘ˆ‘ŽŽ‘™‹‰•–‹—Žƒ–‹‘™‹–Š ƒ†‹†ƒ–‡‡’‹–‘’‡ȋΨ’”‘Ž‹ˆ‡”ƒ–‹‰ͺ ‡ŽŽ•ȌǣΪǡ’‘•‹–‹˜‡”‡•’‘•‡•‡š Ž—•‹˜‡Ž› Ϊ Ϊ ˆ‘—†‹ Ǧʹ ȋŠ‡–‡”‘œ›‰‘—•Ȍ†‘‘”•ǤŠ‡Ψ’”‘Ž‹ˆ‡”ƒ–‹‰ͺ ‡ŽŽ•™ƒ•εȏ‡†‹—Ϊ͵ȗȐ‹–™ƒ• ‘•‹†‡”‡†’‘•‹–‹˜‡Ǥ a b c.    ͹Ͳ.

(109) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. ƒ„Ž‡ ͵† ’”‡•‡–• –Š‡ ——Žƒ–‹˜‡ ͶΪ ƒ† ͺΪ  ‡ŽŽ ”‡•’‘•‡ ’”‘ˆ‹Ž‡• –‘ M. tuberculosis ’”‘–‡‹• ƒ† ”‡•’‡ –‹˜‡ ’‡’–‹†‡• ƒ‘‰  ”‡•’‘•‹˜‡ǡ Š‡–‡”‘œ›‰‘—• Ǧ͵Ϊƒ† ǦʹΪ†‘‘”•Ǥ ‡š–™‡ˆ—”–Š‡”ƒƒŽ›œ‡†–Š‡M.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡†’‡’–‹†‡‡’‹–‘’‡• ‹–Š‡ ‘–‡š–‘ˆ Ǧ͵ƒ† Ǧʹ’”‡•‡–ƒ–‹‘Ǥ ‹”•–ǡ™‡‡šƒ‹‡†”‡•’‘•‡•–‘ ‘–”‘Žƒ–‹‰‡•ǡƒ•ƒ„‘˜‡Ǥ

(110) †‡‡†ǡ ǦʹΪ†‘‘”•”‡ ‘‰‹œ‡†‰ͺͷ’‡’–‹†‡•ͳ͵ǡͳͷǡ ͳ͸ǡʹͲǡƒ†˜ʹͲ͵ͳ ’‡’–‹†‡•ͳͲǡͳʹǡ™Š‹ ŠƒŽŽ ‘–ƒ‹‡†’”‡˜‹‘—•Ž›’—„Ž‹•Š‡† Ǧ ȗͲʹͲͳ ”‡•–”‹ –‡† ‡’‹–‘’‡•Ǥ ‹‹Žƒ”Ž›ǡ ˜ʹͲ͵ͳ  ’‡’–‹†‡ Ͷ –Šƒ– ƒ””‹‡• ƒ Ǧ͵ ‡’‹–‘’‡™ƒ•‹†‡‡†”‡ ‘‰‹œ‡†„› Ǧ͵Ϊ’‘•‹–‹˜‡†‘‘”•ȋ ‹‰—”‡ͳƒ†ƒ„Ž‡͵†Ȍǡ –Š—• ‘ˆ‹”‹‰ ‘ ‡ ‘”‡ –Šƒ– –Š‡ ‡–Š‘† ™‡ —•‡ Š‡”‡ ‹• ƒ„Ž‡ –‘ ‹†‡–‹ˆ› ’‡’–‹†‡ •’‡ ‹ˆ‹ ”‡•’‘•‡•Ǥ •—„•–ƒ–‹ƒŽ—„‡”‘ˆ’‡’–‹†‡•™ƒ•”‡ ‘‰‹œ‡†„›–Š‡•‡Ž‡ –‡† Ǧ͵Ϊƒ† Ǧ ʹΪ †‘‘”•Ǥ Š‡•‡ ’‡’–‹†‡• ™‡”‡ ‡š– •—„Œ‡ –‡† –‘ ˆ—”–Š‡” ‹ •‹Ž‹ ‘ ‹‹ƒŽ ‡’‹–‘’‡ ‘–‹ˆ •‡ƒ” Š‡• ȋʹ͸Ȍ ‹ –Š‡ ‘–‡š– ‘ˆ Ǧʹ ‘” Ǧ͵ –‘ •‡Ž‡ – –Š‡ ’”‡ ‹•‡ ‡’‹–‘’‡• ‡„‡††‡† ‹ –Š‡ ”‡ ‘‰‹œ‡† ʹͲǦ‡”•Ǥ ‡•’‹–‡ –Š‡ Ž‹‹–‡† —„‡” ‘ˆ ‹†‹˜‹†—ƒŽ• ‹˜‡•–‹‰ƒ–‡†ǡ ™‡ ™‡”‡ ƒ„Ž‡ –‘ ‹†‡–‹ˆ› ͳͶ M. tuberculosis ƒ†‹†ƒ–‡ ‘• ”‡‰—Ž‘ ‡ ‘†‡† Ǧ͵ ’”‡•‡–‡† ‡’‹–‘’‡• ȋƒ„Ž‡ ͶƒȌ ƒ† ʹͷ M. tuberculosis ƒ†‹†ƒ–‡‘•”‡‰—Ž‘‡ ‘†‡† Ǧʹ’”‡•‡–‡†‡’‹–‘’‡•ȋƒ„Ž‡Ͷ„ȌǤ  InvitroverificationofcandidateM.tuberculosisDosRregulonencodedepitopes inthecontextofHLADR3 ŽŽˆ‘—”–‡‡ ƒ†‹†ƒ–‡ Ǧ͵M.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡†‡’‹–‘’‡•™‡”‡ ‡š– •›–Š‡•‹œ‡† ƒ• ͳͷǦ‡” ’‡’–‹†‡• ȋƒ„Ž‡ ͶƒȌǡ ƒ† ƒ••‡••‡† ˆ‘” ”‡ ‘‰‹–‹‘ „›  ˆ”‘ –™‘ ‡™Ž› –‡•–‡†  ”‡•’‘•‹˜‡ †‘‘”• ȋ͓ͳͺǣ Ǧ͵ǡ ͷǢ ͓ͳͻǣ Ǧ ͵ǡ ʹȌǡ „‘–Š ‘ˆ ™Š‹ Š ™‡”‡ ‘– ‹ Ž—†‡† ‹ ƒ„Ž‡ ʹǤ ‘–Š †‘‘”• ™‡”‡ ˆ‘—† –‘ ”‡ ‘‰‹œ‡ƒ•—„•–ƒ–‹ƒŽ—„‡”‘ˆ–Š‡•‡M.tuberculosis ƒ†‹†ƒ–‡ Ǧ͵’”‡•‡–‡† ‡’‹–‘’‡• ȋͳͲ ƒ† Ͷǡ ”‡•’‡ –‹˜‡Ž›Ȍ ƒ• •Š‘™ ‹ ‹‰—”‡ ʹƒǡ ™Š‡”‡ƒ• › ‘„ƒ –‡”‹— ƒÃ˜‡ †‘‘”• ʹͲ ȋ Ǧͳǡ ͵Ȍ ƒ† ʹͳ ȋ Ǧ͵ǡ ͹Ȍ †‹† ‘– ”‡ ‘‰‹œ‡ ƒ› ‘ˆ –Š‡ ‡’‹–‘’‡•Ǥ‘•–‹’‘”–ƒ–Ž›ǡ–Š‡•‡”‡•—Ž–••Š‘™–Šƒ–”‡•’‘•‹˜‡‡••–‘M.tuberculosis ƒ†‹†ƒ–‡ Ǧ͵ ‡’‹–‘’‡• ƒ”‡ Ž‡ƒ”Ž› ‘„•‡”˜‡† ‹ ‹†‹˜‹†—ƒŽ• ‡š’‘•‡†Ȁ‹ˆ‡ –‡† ™‹–Š› ‘„ƒ –‡”‹ƒ„—–‘–‹› ‘„ƒ –‡”‹ƒƒÃ˜‡ǡ‡‰ƒ–‹˜‡†‘‘”•Ǥ ‡š–ǡ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ƒ† •‡Ž‡ –‡† ‡’‹–‘’‡• ™‡”‡ ’”‡•‡–‡†–‘ƒ Ǧ͵ΪͶΪ ‡ŽŽŽ‹‡ȋȌ™Š‹ ŠŠƒ†„‡‡‰‡‡”ƒ–‡†ˆ”‘ ‘ˆ ƒ –”‡ƒ–‡†  ’ƒ–‹‡– ȋ Ǧ͵ǡ ͳͷȌ „› •–‹—Žƒ–‹‘ ™‹–Š M. tuberculosis Ž›•ƒ–‡Ǥ –‹‰‡• ™‡”‡ ’”‡•‡–‡† ˜‹ƒ ȋŠ‘‘œ›‰‘—•Ȍ Ǧ͵ ƒ– Š‡† ‘” ‹•ƒ– Š‡† Ǥ ‘” ƒŽŽ ƒ–‹‰‡•ǡ ‡š ‡’– ˜ͳ͹͵͵ ǡ „‘–Š ’”‘–‡‹ ƒ† ’‡’–‹†‡ ”‡•’‘•‡• ‘—Ž† „‡ ‡ƒ•—”‡†Ǥ ‘”˜ͳ͹͵͵ ”‡•’‘•‡•™‡”‡•‡‡ƒ‰ƒ‹•–’‡’–‹†‡Ǧ‡’‹–‘’‡•˜ͳ͹͵͵ ’ͳͷǦʹͻ ƒ†˜ͳ͹͵͵ ’ͳͶͳǦͳͷ͸Ǥ Š‹•”‡•—Ž–‹• ‘’ƒ–‹„Ž‡™‹–Š–Š‡‘–‹‘–Šƒ–’‡’–‹†‡•–‹—Žƒ–‹‘ ƒ ˜‹•—ƒŽ‹œ‡ ƒŽ•‘ ƒ Dz•—„†‘‹ƒ–dz ‡’‹–‘’‡ ”‡’‡”–‘‹”‡ ™Š‹ Š ƒ› ‘– „‡ ˜‹•‹„Ž‡ ˆ‘ŽŽ‘™‹‰ •—„‘’–‹ƒŽ •–‹—Žƒ–‹‘ ™‹–Š ™Š‘Ž‡ ’”‘–‡‹Ǥ ‘–ƒ„Ž›ǡ ‘ ”‡•’‘•‡• ™‡”‡ ˆ‘—† –‘ ’‡’–‹†‡Ǧ‡’‹–‘’‡ ˜ͳ͹͵͵ ’ͳͳǦʹͷ †‡•’‹–‡ ‹–• Žƒ”‰‡ ‘˜‡”Žƒ’ ™‹–Š ˜ͳ͹͵͵ ’ͳͷǦʹͻǢ –Š‹• ’‘‹–• –‘ƒ Ǧ–‡”‹ƒŽ Ž‘ ƒ–‹‘ ‘ˆ ‡••‡–‹ƒŽ ”‡•‹†—‡• ‹˜‘Ž˜‡† ‹‡‹–Š‡” Ǧ͵ „‹†‹‰ƒ†Ȁ‘”Ž‹‰ƒ–‹‘Ǥ

(111)  –Š‹• ǡ ”‡•’‘•‡• –‘ ͳͳ ‘—– ‘ˆ –Š‡ ͳͶ M. tuberculosis ƒ†‹†ƒ–‡ Ǧ͵ ’”‡•‡–‡† ‡’‹–‘’‡• ™‡”‡ ‘„•‡”˜‡†Ǥ ‘ ‘” ˜‡”› Ž‘™ ”‡•’‘•‡• ™‡”‡ ‘„•‡”˜‡† ™Š‡ ƒ–‹‰‡• ‘” ’‡’–‹†‡• ™‡”‡ ’”‡•‡–‡† ˜‹ƒ Ǧ͵ ‹•ƒ– Š‡†  ȋ ‹‰—”‡ ʹ„Ǧ‡ȌǤ. ͹ͳ.

(112) Chapter3. 0. p3 16 -33. Rv 17 33 c Rv p1 517 33 29 c Rv p1 117 33 25 c Rv p1 41 20 -15 29 6 c Rv p3 5-4 20 9 29 c Rv p5 120 29 65 c Rv p1 14 20 -12 29 8 c Rv p1 94 20 -20 29 8 c Rv p2 31 20 -24 29 5 c Rv p2 52 20 -26 31 6 c Rv p4 -18 20 31 c Rv p1 25 26 -13 27 9 c Rv p9 3-1 26 08 27 c Rv p1 86 26 -20 27 0 c. Ž› ˆ‘” ’‡’–‹†‡Ǧ‡’‹–‘’‡ ˜ʹ͸ʹ͹ ’͵ͳ͸Ǧ͵͵Ͳ ƒ ”‡ƒ‹‹‰ ”‡•’‘•‡ǡ ƒŽ„‡‹– •–”‘‰Ž› ”‡†— ‡†ǡ ™ƒ• ‘„•‡”˜‡† ™Š‡ ’”‡•‡–‡† „› Ǧ͵ ‹•ƒ– Š‡† Ǥ Š‹• ‘—Ž† ‹†‹ ƒ–‡ –Šƒ– –Š‹• ’‡’–‹†‡ ‹‰Š– „‡ ’”‡•‡–‡† ƒŽ•‘ „› ‘–Š‡” Ǧ Žƒ••

(113)

(114)  ‘Ž‡ —Ž‡• –Šƒ Ǧ͵Ǥ  ƒ   noPBMCresponse  donor18 PPD positivePBMCresponse no PBMCresponse donor19  donor20  PPD positivePBMCresponse donor21   † „ †            ‡ HLADR3candidate epitope:TCLtestedwith  HLADR3matchedAPC  HLADR3candidate  epitope:TCLtestedwith HLADR3mismatchedAPC         JresponsesinPBMCand  Figure2.RecognitionofcandidateHLADR3epitopesbyassessmentofIFN inCD4+Tcelllines.PBMCfromPPD+HLADR3+(heterozygous)donors(donor18and19)bothrecognized  asubstantialnumberofcandidateHLADR3epitopes,incontrasttoPPDHLADR3+(heterozygous)donors  (blackbox,response,whitebox,noresponse)(a).ImmunodominantM.tuberculosisdosRantigensandtheir  respectivecandidateHLADR3epitopesweretested,usingaCD4+ M.tuberculosislysateinducedTcellline  (TCL) with HLADR3 matched (homozygous) APC (black bars) (be). For Rv2029c (d), Rv2031c (c) and Rv2627c (e) responses to both protein and candidate epitopes were observed. For Rv1733c (b) only  responses to its respective candidate epitopes were observed (see text for discussion).  When the TCL was  tested with DR3 mismatched APC (white bars), no or strongly reduced responses were observed (be). An  IFNJresponse t100pg/mlwasconsideredpositiveforPBMCandforM.tuberculosisantigenspecificTcell  lineswhenIFNJresponsewast50pg/ml. +. . ͹ʹ.

(115) CD4+andCD8+TcellresponsestoM.tuberculosisDosRantigens. ‘ˆ‹”‹‰–Š‡ƒ„‘˜‡‘„•‡”˜ƒ–‹‘•ǡ”‡•’‘•‡•–‘–Š‡Ž‘‰‡”ʹͲǦ‡”’‡’–‹†‡•‘ˆ–Š‡M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ƒ–‹‰‡• ‘–ƒ‹‹‰ –Š‡ ͳͷǦ‡”‹  Ǧ͵ ƒ†‹†ƒ–‡‡’‹–‘’‡•™‡”‡ƒŽ•‘‘„•‡”˜‡†ȋ†ƒ–ƒ‘–•Š‘™ȌǤŠ—•ǡ–ƒ‡–‘‰‡–Š‡”–Š‡•‡ ”‡•—Ž–••–”‘‰Ž›•—‰‰‡•––Šƒ–ƒ•‡”‹‡•‘ˆM.tuberculosis‘•”‡‰—Ž‘‡ ‘†‡†‡’‹–‘’‡• ‹•’”‡•‡–‡†–‘ͶΪ ‡ŽŽ•„› Ǧ͵‘Ž‡ —Ž‡•Ǥ   InvitroverificationofcandidateM.tuberculosisDosRregulonencodedepitopes inthecontextofHLAA*0201 ƒ‹‰ ƒ •‹‹Žƒ” ƒ’’”‘ƒ Šǡ ʹͷ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ǦȗͲʹͲͳ ”‡•–”‹ –‡† ƒ†‹†ƒ–‡ͻǦ‡”‹ ‡’‹–‘’‡•™‡”‡•›–Š‡•‹œ‡†Ǥ ‹”•–ǡ–Š‡‹”„‹†‹‰–‘ Ǧʹ ™ƒ•‡ƒ•—”‡†ȋƒ„Ž‡Ͷ„ȌǤ‡˜‡”ƒŽ‘ˆ–Š‡‹•‹Ž‹ ‘’”‡†‹ –‡† ƒ†‹†ƒ–‡ Ǧʹ‡’‹–‘’‡• Šƒ† ‹–‡”‡†‹ƒ–‡ ‘” Š‹‰Š „‹†‹‰ ƒˆˆ‹‹–› ȋ

(116) ͷͲ δͳͲ ɊȌ ˆ‘” ǦȗͲʹͲͳ ‘Ž‡ —Ž‡•Ǥ –Š‡” ƒ†‹†ƒ–‡ Ǧʹ‡’‹–‘’‡•ǡ†‡•’‹–‡Š‹‰Š’”‡†‹ –‹‘• ‘”‡•ƒ†‹˜‹–”‘ͺΪ ‡ŽŽ ”‡ ‘‰‹–‹‘ ‘ˆ –Š‡ ‘””‡•’‘†‹‰ ʹͲǦ‡” ’‡’–‹†‡ǡ Šƒ† Ž‘™ ȋ

(117) ͷͲεͳͲ ɊȌ ‘” ‘ ‡ƒ•—”ƒ„Ž‡ȋ

(118) ͷͲεͳͲͲɊȌƒˆˆ‹‹–›ˆ‘” ǦȗͲʹͲͳ‘Ž‡ —Ž‡•Ǥ  a  Figure 3. Cytotolytic activity of a  CD8+ polyclonal T cell line  against the HLAA2 restricted M. tuberculosis epitope Rv1733cp181  189. The results show specific  recognition of peptide epitope  Rv1733cp181189 byCD8+Tcellswhen peptide was loaded onto EBVBLCL  JY target cells (compared to  medium control). CD8+ T cells also  recognizedEBVBLCLJYtargetcells  that had been transduced with an expression plasmid encoding  Rv1733c, thus facilitating  endogenous expression and  presentation of Rv1733c in the b context of HLAA*0201. In contrast  EBVBLCL JY target cells which  were transduced with an empty  plasmid (a) were killed to a much  lesser extent. Specific lysis of EBV BLCL JY target cells loaded with  Rv1733cp181189 could be inhibited  partly by addition of antiHLA  classI but not antiHLAclassII antibody (b). The % specific lysis  wascalculatedasfollows:[(release  spontaneous release)/ (maximum  releasespontaneous release)] x  100%.     . ͹͵.

(119) Chapter3. ŽŽʹͷ ƒ†‹†ƒ–‡ Ǧʹ‡’‹–‘’‡•™‡”‡–‡•–‡†‹ƒŽ›’Š‘ ›–‡•–‹—Žƒ–‹‘–‡•–‹ͳͳ ǦʹΪ ƒ†ͷ ǦʹǦ‹†‹˜‹†—ƒŽ•ȋΪ„Ž‘‘†„ƒ†‘‘”•ȋ‡‹–Š‡”ˆ”‘ƒ„Ž‡ʹ‘” ‡™Ž› –‡•–‡†Ȍǡ  ‘˜‡”–‡”• ‘”  ’ƒ–‹‡–•Ȍ –‘ ‡šƒ‹‡ –Š‡‹” ”‡ ‘‰‹–‹‘ ‹ –Š‡ ‘–‡š–‘ˆ Ǧʹ‘Ž‡ —Ž‡•Ǥ ‹˜‡ Ǧʹ ƒ†‹†ƒ–‡’‡’–‹†‡•™‡”‡”‡ ‘‰‹œ‡†•‘Ž‡Ž› „› ǦʹΪ „—– ‘– ǦʹǦ †‘‘”• ƒ•ƒ••‡••‡† „›   ’”‘Ž‹ˆ‡”ƒ–‹‘ ȋƒ„Ž‡ Ͷ„ȌǤ  ’‘Ž› Ž‘ƒŽͺΪ ‡ŽŽŽ‹‡™ƒ•‰‡‡”ƒ–‡†–‘‡š’Ž‘”‡’‘••‹„Ž‡ˆ— –‹‘ƒŽ’”‘’‡”–‹‡•‘ˆ ͺΪ  ‡ŽŽ•†‹”‡ –‡† –‘ –Š‡•‡ M. tuberculosis ‘• ”‡‰—Ž‘ ‡ ‘†‡† ‡’‹–‘’‡•Ǥ  ˆ”‘ ƒ  ’ƒ–‹‡– ȋ Ǧʹǡ Ǧ͵ǡ ƒ† ǦͳͳȌ ™‡”‡ •–‹—Žƒ–‡† ™‹–Š ƒ ‘„‹ƒ–‹‘ ‘ˆ –Š‡ ƒ†‹†ƒ–‡ Ǧʹ ”‡•–”‹ –‡† ‡’‹–‘’‡• ˜ͳ͹͵͵ ’ͳͺͳǦͳͺͻǡ ˜ʹͲ͵ͳ ’ͻ͸ǦͳͲͶ ƒ† ˜ʹ͸ʹ͹ ’ͳͷͳǦͳͷͻǤ ŽŽ –Š”‡‡ ’‡’–‹†‡• ™‡”‡ ”‡ ‘‰‹œ‡† „› –Š‹• †‘‘” ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ ˆˆ‡ –‘” ͺΪ  ‡ŽŽ• •Š‘™‡† •’‡ ‹ˆ‹  ’‡’–‹†‡ †‡’‡†‡– Ž›•‹• ‘ˆǦ  –ƒ”‰‡– ‡ŽŽ• ȋ ǦȗͲʹͲͳȌ ™Š‡ ’—Ž•‡† ™‹–Š ’‡’–‹†‡ ˜ͳ͹͵͵ ’ͳͺͳǦͳͺͻ ȋ„—– ‘– ™‹–Š ˜ʹ͸ʹ͹ ’ͳͷͳǦͳͷͻ‘”˜ʹͲ͵ͳ ’ͻ͸ǦͳͲͶȌǤ‹ŽŽ‹‰™ƒ•‘„•‡”˜‡†ƒ–†‹ˆˆ‡”‡–‡ˆˆ‡ –‘”Ǧ–‘Ǧ–ƒ”‰‡– ”ƒ–‹‘• ȋǣ Ȍ ȋ ‹‰—”‡ ͵ƒȌǤ • ‡š’‡ –‡†ǡ –Š‡ —•‡ ‘ˆ ƒŽŽ‘‰‡‡‹  Ǧ –ƒ”‰‡– ‡ŽŽ• ‹ ‘„‹ƒ–‹‘ ™‹–Š ’‘Ž› Ž‘ƒŽ ‡ˆˆ‡ –‘” ‡ŽŽ• ”‡•—Ž–‡† ‹˜ƒ”‹ƒ„Ž› ‹ •‘‡ „ƒ ‰”‘—† ƒ –‹˜‹–›ǡ‡˜‡‹•— Š•Š‘”––‡”ƒ••ƒ›•ȋͷǦ͵ͷΨŽ›•‹•‘ˆ ‘–”‘Ž–ƒ”‰‡–•ȌǤ‡˜‡”–Š‡Ž‡••ǡ ’‡’–‹†‡ †‡’‡†‡– Ž›•‹• ™ƒ• Ž‡ƒ”Ž› ‡˜‹†‡–Ǥ ͺΪ  ‡ŽŽ• ƒŽ•‘ ”‡ ‘‰‹œ‡† ƒ† Ž›•‡† Ǧ –ƒ”‰‡– ‡ŽŽ•–Šƒ–Šƒ†„‡‡–”ƒ•†— ‡†™‹–Šƒ’Žƒ•‹†‡ ‘†‹‰˜ͳ͹͵͵ ǡ ™Š‹Ž‡ ‘– ”‡ ‘‰‹œ‹‰ Ǧ  –ƒ”‰‡– ‡ŽŽ• •‹‹Žƒ”Ž› –”ƒ•†— ‡† ™‹–Š ‘–”‘Ž ’Žƒ•‹†ȋ ‹‰—”‡͵ƒȌǤ ’‡ ‹ˆ‹  ’‡’–‹†‡ †‡’‡†‡– Ž›•‹• ‘ˆ Ǧ  –ƒ”‰‡– ‡ŽŽ• Ž‘ƒ†‡† ™‹–Š Ǧ ƒ†‹†ƒ–‡‡’‹–‘’‡•˜ͳ͹͵͵ ’ͳͺͳǦͳͺͻ ‘—Ž†„‡‹Š‹„‹–‡†’ƒ”–Ž›ȋ̱͵ͲΨȌ„›–Š‡ƒ††‹–‹‘ ‘ˆ ƒ ’ƒ ƒ–‹Ǧ Ǧ Žƒ••

(120)  ƒ–‹„‘†› ™Š‡”‡ƒ• ‘ †‡ ”‡ƒ•‡ ‹ Ž›•‹• ™ƒ• ‘„•‡”˜‡† ˆ‘ŽŽ‘™‹‰ƒ††‹–‹‘‘ˆ’ƒƒ–‹Ǧ Ǧ Žƒ••

(121)

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