CD8α+β− intestinal intraepithelial lymphocytes in T cell receptor transgenic mice are positively selected by high affinity ligands

23 ~ne 1 ~ 7 - Poster p ~ s e n t a t i o ~

Un~~l

wit~n a month after flint detection. We observed a similar CD4dU"CD8b~g~ DP ~mphocyte subpopul~on ~ a p ~ e ~ on ~munosuppressive ~erapy a~er renal transplan~tion dudng asym~oma~c cytomegalo~ms ~ M ~ ma~va~on. Me~od~ Peripheral ~ood was drawn be~re and ~ seve~ ~me inte~a~ a~er ran~ tranaplan~tion. CMV se~ogy and CMV c~tura ~ udne and ~ood was pe#ormed mpeated~ FACS-ana~sis was pe~ormed on peripheral ~ood monon~lem cel~ u~ng ~rect~ co~ugated fluomsce~ monoc~n~ a~bodias. A 1% pamformaldehyde ~ o n and ~1% saponin permea~lisation procedure was performed for d~e~on ~ ~racel~lar a~gens. Results: CD4dullcD8bdgM DP c~s were pmse~ ~ a sm~ amou~ bea m transplan~on (1 ~ 2%); showed an ~crease to 5% dudng CMV raa~a~on and p e ~ e d ~r m ~a~ 8 m o n ~ These cel~ appeared ~ be >98% CD~CD~TCR~p*CDIa-CD16- and 94% CD~. They were also 92% granwme B+ and 83% CD5~. The calls lacked a CD45RAb~g~CD11adu"n~ve subs~ wh~h was chara~e~s~cally present wit~n ~e CD8~g~ ~n~e po~ve sube~ The C D ~ C D ~ r~t DP cel~ ~d n= show expras~on ~ the e a ~ activa~on molecule CD69 nor ~ the IL-2 race,or =Chin CD25. HLA~R was expressed by 35% ~ CD4dU~=CD8b~ DP cel~ Conclusion: Expan~on ~ the C D ~ C D 8 ~ DP c~s ~ t~s p ~ e ~ rem~ned detecta~e ~r ~ ~ast 8 mo~h¢ These ce~s am memow/effe~or TCR=~* Tolymphocytas, wh~h do n~ express ma~em ~ mce~ a ~ o n l i ~ CD25 and CD6~ The expression ~ granwme B and CD57 by ~ese calls, w~ch am ma~em f ~ differen~ated cytoto~c T lymphocytes, indicate ~ ~ey do n~ belong ~ the most m a i m thymic T-lymphocytesubset, as was suggested pmvious~.

[

I and/or somatic mutagensei$?

Kh.I. Istamov ~, G.V. Tchempnev2, ~1. Krasil~kova 3, Z.M. Nechoroshkova3. 1N~ona/Research C e n ~ Institute of Immun~o~, Mosco~ Russia, 2Repu~ican Medical Diagnostic Cente~ Kaza~ Russia, 3 Repu~ican Clinical Hospita~ Kaza~ Russia Introduction: DNA damage in immunologically a~ive cells may result in acq~red immunodefi~ancy (Kamulov A.V et eL, 1996). ~ seems ~emfom masona~e ~ =udy the key cell surface m~ec~as ~ . CD3/I"CR) whi~ simul~necu~y inve~iga~ng ~e rate ~ muta~ons ~ the respective gene loci. An ~ m a t i v e , eas~r approach mig~ be measuring the frequency ~ de~=ive vada~ cel~ expressing ~e appmpd=e =~red pheno~pe. The objective was ~ s~dy the ~equency ~ varia~ CD3-CD4+ T lymphocytes among CD3+CD4+ peripheral ~ood cells in relation ~ ag~ underling d~ease and occupa~on~ exposure ~ ~nizing m~ation. M a t e r ~ and Methods: To enumerate CD3-CD4+ defective vada~ ce,s we performed a 3-color flow cytometdc assay (CD3-ECD/CD4-PE/CD8-RTC) u~ng C o ~ r E~cs XL and Mu~-Q-pmp ~ation. The ~mphoc~e f r a ~ n was gated by ~rwa~ and dght-an~e ~g~ sca~er and a window ~r vadant CD3-CD4+ cells was set up as specBed by Kyoizumi~ etal. ~992). Subjects ~ ~e ~udy ~duded (1) patien~ with atopic disease ~ = 10 wome~ and (2) medical s~ff ~ Radi~ogy Department ~ = 19 women. Reault~: The frequency ~ vada~ CD3-CD4+ cel~ (V~ ~ heaRhy donom vaded kom 2 ~ 6 x 10-4 among the CD3+CD4+ cel~ ~ ~e peripheral ~ood wh~h does n~ c o ~ m ~ ~e mean Vf = 2.5 x 10-4 o ~ n e d by Kyoizumi ~ eL al. ~992). The mean =anda~ de~ation ~ n=ural Iog~ransformed vadant frequency ~Vf [×10 -4] (used because ~e raw Vf v=ues were n~ normally ~stdb~e~ ~ ato~c patients was significantly higher ~an ~ ~ medical staff ~ ~s Radi~ogy Depa~me~ (3.18 ± 1.07 vs. 1~7 ~ ~73, p < ~0~. A moderate pos~ve correlation (r = 0.5~ was detected between ~e CD3-CD4+ Vf end absolve CD8+ count. The CD3-CD4+ Vf did not correlate with the age and length ~ sewice ~ ~e medical ~aff ~ ~e R a ~ o g y Depa~me~ w~ch mig~ be due ~ the sta~s similad~ ~ ag~ Conclusion: Determining the frequency ~ CD3 de~ctive vadan~ mig~ be u s ~ as a potenti~ ~ c a t o r ~ T-cell immunodefidency, as we~ as somatic mumgene~¢ To ved~ ~e T cell ra~er ~an monocyte ~neage ~ CD3-CD4+ vada~ cells ~co~r CD~CD4/CD14 ~ n i n g ~ needed.

I P.2.03.03 ] TCDao=+~-ce " receptorintestinal intrsepithellal mice are positively selected

in

by high affinity .gands

S~phen 1 Simpson ~ , Andrew M~lor 3, Cox Te~orst ~, Susumu Tonegawa~, Chdstiaan Levelt 4. ~Di~sion of Immunology, Be~ Isra~ and Deaconess Medical Centre, Harvard Medical School, Boston,/idA, USA, 2Education and Reseamh Centre, St Vincen~s Hospi~L Du~in, Irelan~ 4Massachusetts Institute ~r Technolog~ Cambridge, Massachusetts, USA, 3Univemi~ Medical Centre, Augusta, Georgia, USA Introduction: TCR=~* i~raepitheli~ lymphocytes ~ ~e mudne sm~l intestine ~ compdse two pmdomina~ populations ~ T cells, chara~edsed by ~e

~ m p ~ t e su~e~

99

expms~on ~ ~e CD8~ chin; CD8~+p+ and CD8~*~- gE~ A number ~ =udies have demon=rated that CD8~*p- ~ wild type mice c o ~ n ~gnBca~ numbem ~ T ce~s which express TCR spedfic~es ~r self an~gens and ~ h ~ stu~es have provided ev~ance ~ sugge= th= CD8~*~- ilEL may ~ fa~ be pos~vely s~ected by an~gens for which their recep~m have a high affini~ We have investigated this qua=ion ~ two ~ffemnt lines ~ TCR transgenic mic~ F5 mice and BM3 mice. F5 mice express a ~ansganic TCR specific for ~e influenza n u d e o p m ~ ~ ~e conte~ ~ H~D b m ~ e c ~ In ~ese anim=s ~ymocytas am positive~ selected ~ H-2~ mice b~ are deleted ~ the presence ~ injected sdub~ peptide (Mam~a~ ~ ~ 1992. PNA~ 8~ 1134~. BM3 transgenic mice express TCR chins specific for ~e H-2Kb molecule. Thymocytes am deleted by ~ ~gand but are pos~ve~ selected by H-2Kk molecules. Methodm ~ order ~ examine ilEL developme~ ~ ~e absence ~ endog~ nously expressed TCR, transgenic mice crossed with RAGnu" mice were used. IEL hawested from ~e small intestines ~ mice were exam~ed by ~me c~our flow c~omet~ Results: In F5 a n i m ~ the gEL compartme~ confined ~gn~cam percenF ages ~ CD8~z*~*, b~ CD8~*~- ,EL were ~mo~ c o m ~ e ~ absent. ~mi~d~ in H ~ B M 3 mice CD8~*p*, b~ n~ CD8~*~- ilEL were present. To de,rinse wh~her CD8~*~- cou~ develop in the presence ~ ~ r high affini~ ligan~ we examined F5 and BM3 mice which expressed ~ r respective deleting ~gands. ~ F5 mice this was achieved by ~e co-expression ~ a transgene encoding ~e NP 366-374 peptide under a class I MHC promoten In ~ese a~m=s CDa~*~cal~ were pmse~ b~ CD8~*p* were absent. In H~b-BM3 mice, a ~gn~ce~ peme~age ~ CD8~+#-, b~ ~w CD8~+~+ ,EL were d~ected. Furthermore, ~ situ quan~tative analysis ~ numbem ~ ilEL using immunmh~tochemical ==n~g, reve~ed up ~ six ~mas ~e number ~ CD8~* ilEL ~ F5 peptide transgan~ mice compared with F5 mice ~ = d~ n~ express ~e pep~detransgene. Con~ua~n: These da~ sugge= th= CD8~*p- IEL a~ively req~m ~e presence ~ Ngh a f f i ~ I~and for developme~ within the intes~nal epith~ium, whilst CD8~*~* ~EL fail ~ develop pmsumab~ due ~ ~ r delet~n from ~e ilEL mpedoire.

I P.2.03.041 methods toCompar~onthechoose of thedifferentmoatPermeabigzatifor Onauithe tabledetect the intracellular receptors E. ~ h ~ 1, ~ Be~i 2, G~ Lus~ik 2, ~ Szab~ *. ~Natl Blood Center Budape$~ Hunga~ 2Medical Univ Pdcs, Hunga~ Introdu~lon: ~ seems more and morn importa~ ~ examine the intrace,~ar receptom witch the cel~ (Both ~e i~m c~o~asm~ or ~ a nude~ar recep~m). The ~vestiga~on ~ these race,ore am impo~a~ n~ only for ~e bas~ sconce b~ ~ co~d be ve~ impoRa~ from the clinical aspe~ too. ~ example ~ separate the TH1/I'H2 cell~. B~ the effe~ive examina~on~ ~e intmce,ular race,ore depends from the ap~ied method ~ permeapil~ation. M~er~Is and Method~ The ~ffem~ commemi~ permea~flza~on me~ods, ava,a~e on the manet were compam~ in norm~ ~ood donom, and patien~ ~ leukaemia. The permeabilization kits were ~e ~llowing: Cytoperm from SEROTEC, ~X and PERM from AN-DER-GRUB BioREsea~h GmbH, C~o Stun G ~ Stop kit From Ph~ Mingen, Modred C~o Stain kR (Hungaw) and Permea~a~on ~t from Becton D~nson. For ~e intracellular s ~ g GCR-FWC and and 1~2-FITC, for the cellsurface staining CD3-PerC~ CD~PE, CD4-PerCR CD4-PE were ap~od using wh~e ~ood and sapamted monon~ ~ear cel~ Conclusion:To select the be= me~od for ~e i~racellulm ~ves~gation can help analyze not on~ the intra c~oplasm~, but the intra nucleolar receptom too. I

I ~ 2 . 0 3 . 0 5 1 Human

peflpher~ ~ T o e l ~ ~ HIV ~ f e c t l o n

~ Medina, ~ Paunesc~ ~ C o ~ M. Cucum~ M. Sedan. Immunology Department Medical Unive~it~, Timisoara, Romania I~rodu~lon: M ~ ~ u m s ~ ~e immune response ~ HW infect~n such as the ~volvement ~ CD8+ cyt~ox~ T cells and a general s~fl ~wa~s a ThO profi~ were wag documented in the last yearn. Several da~ sugge~ ~gn~ca~ a~erations ~ ~e y~ T cells subs~ ~ d b ~ i o n ~ peripheral ~ood from HIV-infe~ed pemon~ This =udy was aimed ~ invast~ate some fano~pic ~atums ~ the minor popula~on~ ~mula~ng ya T cells ~ HIV-i~ected children. Mater~ls and Methods: Bood samples from 20 HIV-i~ected children ~thout any evidence ~ mycobacted= ~ i o n were assessed ~r ~e expression of CD ma~em by two and ~ree coloum flowc~ometdc analysis on a Lysys II profile. Be~on ~c~nson mouse an~human C~ RTC, PE and PerCP ~b~ed monoc~n~ an~bodies and FACSod floc~ometer were use~ Sambas prap~ rat~ns ~r ~e =orage and dam acquisNons ecco~ed ~ ~e manu~umr~ ~mctions. T cells were gated ~ i n the FSC/SSC features and the ~v~ ~ CD~ CD14 and CD45 expression on the~ sudaces. D ~ o ~ and histogram an~yses were performe~ Reault~: ~ 15 cases the ya T cells were between 10-55% ~ dmula~ng ~mphoc~e¢ 56% ~ ~em ~d express ~e HLA-DR and CD69 a~ivation ma~em