EMS a 1uN Miltenyi Ex. 1029 Page 1
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`duc:2 8 2000
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`Miltenyi Ex. 1029 Page 1
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`

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`
`stereoscopic system
`with reverse correlation
`P Neri, AJParker & CBlakemore
`
`682 Lanthanum-substituted
`bismuth titanate for use in non-
`volatile memories
`BH Park, BSKang, S D Bu,
`699 Top-downsignalfrom
`TWNoh, J Lee
`prefrontalcortex in executive
`& WJoN&V
`control of memory retrieval
`H Tomita, M Ohbayashi,
`685 Two-dimensional charge
`KNakahara,IHasegawa
`transport in self-organized,
`& YMiyashitaN&V
`high-mobility conjugated
`polymers
`703 L-type calcium channels and
`H Sirringhaus, PJBrown,
`GSK-3 regulate the activity of
`RH Friend, MMNielsen,
`NF-Atc4 in hippocampal neurons
`KBechgaard, :
`
`
`BMWLangeveld-Voss, A] H IA Greaf, PG Mermelstein,
`Spiering, RA JJanssen,
`KStankunas, JR Neilson,
`EWMeijer, PHerwig
`KDeisseroth, R WTsien
`& DM de Leeuw
`e& GR Crabtree
`
`MR Egmond, NDeeker
`& BWDijkstra
`721 Thereaction cycle of
`isopenicillin Nsynthase
`observedby X-raydiffraction
`NIBurzlaff, PJ Rutledge,
`TJ Clifton, C M H Hensgens,
`MPickford, R MAdlington,
`PL Roach &JE Baldwin
`
`
`
`
`
`688 Identifying magma—water
`interaction from the surface
`features of ash particles
`R Bittner, P Dellino
`&B Zimanowski
`
`708 Two subsets of memory
`T lymphocytes with distinct
`homing potentials and
`effector functions
`F Sallusto, D Lenig, R Forster,
`MLipp &A Lanzavecchia N&VV
`
`new on the market
`PPPTPTTTrey
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`725 Genetics
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`classified
`Backpages. Search and Browse this entire section at
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`NATURE] VOL401| 14 OCTOBER1999| www.nature.com
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`Miltenyi Ex. 1029 Page 4
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`

`

` letters to natureST
`
`Two subsets of memory
`T lymphocytes with
`distinct homing potentials
`and effector functions
`Federica Sallusto*, Danielle Lenig*, Reinhold Forster}, Martin Lippt
`& Antonio Lanzavecchia*
`
`1. Mulkey, R. M, Endo,S., Shenolikar, $. & Malenka, R. C. Involvementof a calcineurin/inhibitor-1
`phosphatase cascadein hippocampal long-termdepression. Nature 369, 486-488 (1994),
`2. Lu, ¥.F, Hayashi, Y., Moriwaki, A.,Tomizawa, K. & Matsui, H. FK506, a Ca2+/calmodulin-dependent
`phosphatase inhibitor inhibits the inductionoflong-term potentiationin the rat hippocampus.
`Neurosci. Lett. 205, 103-106 (1996).
`3. Winder,D. G., Mansuy, Il. M., Osman, M., Moallem,T. M. & Kandel, E. R. Genetic and
`pharmacological evidence for a novel, intermediate phase oflong-term potentiation suppressed by
`cakineurin. Cell 92, 25-37(1998).
`4. Mansuy, I. M., Mayford, M., Jacob, B., Kandel, E. R. & Bach, M. E.Restricted and regulated
`‘overexpression reveals calcineurinas a key componentin the transition from short-termtolong-term
`memory. Cell 92, 39-49 (1998).
`5. Klee, C. B.,Crouch, T. H. & Krinks, M. H. Calcineurin: acalcium- and calmodulin-binding proteinof
`the nervous system. Proc. NatlAcad. Sci. USA 76, 6270-6273 (1979).
`6. Flanagan, W. M., Corthesy, B., Bram, R. J. & Crabtree, G. R. Nuclearassociationof a T-cell
`transcriptionfactor blocked by FK-506 andcyclosporin A [see comments]. Nature 352, 803-807
`(1991).
`7. Liu, J. et al. Calcineurinis a common target ofcyclophilin-cyclosporin A and FKBP-FK506
`complexes. Cell 65, 307-815 (1991),
`* Basel Institute for Immunology, Grenzacherstrasse 487, Postfach,
`8. Shaw, J-P. fal, Identificationofa putativeregulatorofearlyT cell activation genes. Science241, 202—
`205 (1988).
`CH-4005 Basel, Switzerland
`+ Max-Delbrueck-Centerfor Molecular Medicine, Robert Rossle Strasse 10,
`9. Clipstone, N. A. & Crabtree, G. R. Identification ofcalcineurin as a key signalling enzyme in T-
`lymphocyte activation, Nature 357, 695-697 (1992)
`13122 Berlin-Buch, Germany
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`10, Hoey, T, Sun, ¥.-L., Williamson, K. &Xu,X. Isolation oftwo new members oftheNF-ATgene family
`and functional characterization ofthe NF-AT proteins. Immunity2, 461-472 (1995),
`11.Crabtree, G. R. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-
`Naive T lymphocytestravel to T-cell areas ofsecondary lymphoid
`AT.Cell 96, 611-614 (1999).
`organsin search ofantigen presented by dendritic cells’*. Once
`12.
`Jain,J. et al. TheT-cell transcription factor NFATp isa substrate for calcineurinandinteractswith Fos
`and Jun, Nature 365, 352-355(1993).
`activated, they proliferate vigorously, generating effector cells
`that can migrate to B-cell areas orto inflamedtissues’. A fraction
`13,Beals, C. R., Clipstone, N. A., Ho, S, N. & Crabtree, G. R. Nuclearlocalization of NF-ATe by a
`calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev. 11, 824-834
`(1997).
`of primed T lymphocytespersists as circulating memory cells that
`can confer protection and give, upon secondary challenge, 2
`14, Timmerman,L. A., Clipstone, N, A., Ho,.N., Northrop, J.P. &Crabtree, G. R. Rapid shuttlingofNF-
`ATin discrimination ofCa2+ signals and immunosuppression. Nature 383, 837-840 (1996).
`qualitatively different and quantitatively enhanced response”.
`The nature of the cells that mediate the different facets of
`15. Beals, C. R, Sheridan, C. M., Turck, C. W., Gardner, P, & Crabtree, G. R. Nuclear export of NF-ATc
`enhanced byglycogen synthase kinase-3. Science 275, 1930-1934 (1997),
`immunological memory remainsunresolved. Here we show that
`16. Chow, C. W., Rincon, M., Cavanagh,J., Dickens, M. & Davis, R. J. Nuclear accumulation ofNFAT4
`expression of CCR7, a chemokinereceptor that controls homing
`opposed bythe JNK signal transduction pathway. Science 278, 1638-1641 (1997),
`17, Zhu,J. et al. Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and
`to secondary lymphoid organs, divides human memoryTcells |
`MEKKI.Cell 93, 851-861 (1998).
`into two functionally distinct subsets. CCR7 memory cells
`18. He, X,Saint-Jeannet, J. P,, Woodgett,J. R., Varmus, H. E. & Dawid, I. B, Glycogen synthase kinase-3
` = -_—_ ce708—_
`
`NATURE| VOL 401|14 OCTOBER 1999|www.nature.com
`Miltenyi Ex. 1029 Page 5
`
`express receptors for migration to inflamed tissues and display
`
`coe reecevecc croc seece cece eee ness eeese ress eeaseeeesecceconoeees
`
`Miltenyi Ex. 1029 Page 5
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`

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`letters to naturees
`
`This migration mediates rapid protective responses and is con-
`express lymph-node homingreceptors and lack immediate effec-
`trolled bythe expressionofdifferentsets ofintegrins and chemokine
`tor function, but efficiently stimulate dendritic cells and differ-
`receptors", However, some memoryTcells must also reach the
`entiate into CCR7effectorcells upon secondary stimulation. The
`lymph nodes to mount secondary proliferative responses. We
`considered whether the two facets of the memory response might
`CCR7* and CCR7_T cells, which we have namedcentral memory
`dependonsubsets of memoryT cells invested with distinct homing
`(Tex) and effector memory (Tpy), differentiate in a step-wise
`andeffector capacities.
`fashion from naive T cells, persist for years after immunization
`Because CCR7 and CD62Lareessential for lymphocyte migration
`and allow a division of labour in the memory response.
`When blood-bornenaive T cells hometo lymph nodes,theyfirst
`to lymph nodes'’, the co-expression of these receptors might
`roll on high endothelial venules using CD62L. This allows the
`distinguish a putative subset of memory T cells that home to
`chemokine receptor CCR7 to engage its ligand SLC, which is
`lymph nodes. Humannaive and memoryTcells can be identified
`bythe reciprocal expression of the CD45RA or CD45R0isoforms'*.
`displayed by endothelial cells. The CCR7—-SLC interaction acti-
`vates integrins that promote firm adhesion and transmigration of
`Staining ofperipheral blood T cells with antibodies to CD45RA
`and CCR7 revealed three subsets of CD4* cells: one naive
`the T cells into the lymph node”. In contrast to naive T cells,
`memory/effectorcells migrate mostly through peripheraltissues",
`CD45RA*CCR7*; and two memory subsets, CD45RACCR7* and
`
`a
`b
`
`Figure 1 CCR7 and CD62Lare co-expressed on a subset of peripheral blood memory
`CD4and CD8*Tcells. CD4* (a, b) and CD8* (c, d) lymphocytes were stained with
`2
`fi
`monoclonal antibodies to CD45RA and CCR7, whichidentified three andfour subsets,
`eA
`respectively. These subsets were sorted and analysedfor the expression of CD62L, and
`the percentageof bright cells is indicated (b, d). Uponserial analysis, the proportion of
`:
`cells in the different compartments was rather stable in the sameindividual, but more
`variable among individuals, the variability being more pronounced in the CD8 than in the
`45%
`CD4 compartment. Comparable results were obtained using two anti-CCR7antibodies
`aa
`(clones 3D12 and 10HS).
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`and CD45RA asin Fig. 1 and tested for thelr capacity to produce IL-2 or IFN-y (c) orwere
`Figure 2 CCR7* and CCR7- memoryTcells display different effector functions. a, b, The
`immediately stained with anti-perforin antibody (green) and counterstained with
`three subsets of CD4* T calls were sorted according to the expression of CCR7 and
`propidium lodide (red) (d). In the CDB* CD45RA* compartment, CCR7 expressionallows
`CD45RA as in Fig. 1 and testedfor their capacity to produce IL-2, IFN-y, IL-4 and IL-5 (a)
`usto discriminate naivecells (1) from effector cells (4) (ref. 26). Comparable results were
`andfor the kinetics of surface CD40L upregulation (b)following polyclonalstimulation.
`obtained in 12 healthy donors.
`¢, d, The four subsets of CD8* T cells were sorted accordingto the expression of CCR7
`
`NATURE|VOL401| 14 OCTOBER 1999|www.nature.com
`
`709
`
`Miltenyi Ex. 1029 Page 6
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`Miltenyi Ex. 1029 Page 6
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`

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`NATURE| VOL 401|14 OCTOBER 1999 |www-nature.com
`Miltenyi Ex. 1029 Page 7_
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` expressed CD62Lto a lower and variable extent (Fig. 1b). Within
`
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`
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`
`cells is dependent on expression of CD40L”, whereas protective
`responses in the tissues are mediated byT cells that produceeffector
`cytokines, such as interferon-y (IFN-y) or interleukin-4 (IL-4), or
`release stored perforin””*, The naive and two memory CD4 subsets
`were sorted and compared for their capacity to produce cytokines
`and upregulate CD40L following stimulation. As shown in Fig.2a,
`both naive T cells and CCR7* memory cells produced IL-2 only. In
`contrast, the CCR7 memory subset produced high levels of IL-4,
`IL-5 and IFN-y and moderately reduced levels of IL-2. Upon
`activation, the extent of CD40L upregulation was comparable in
`the two memory subsets and was higher than in naive T cells;
`however, the kinetics of upregulation were comparable, indicating
`that, unlike tonsil T cells”, circulating memory T cells do not
`contain stored CD40L (Fig. 2b). Rapid production of IFN-y was
`detected in most CCR7, but only a negligible fraction of CCR7*
`
`a
`
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`b
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`18%
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`8
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`6
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`0
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`100
`160
`200
`250
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`T cells x 10°3
`0.3%
`{
`
`Figure 4 CCR7* memory calls show enhanced responsiveness to T-cell receptor
`triggering and potently activate dendritic cells to produceIL-12. a, Proliferative response
`of naive T cells (squares), CCR7* (triangles) and CCR7~ (circles) memory T cells to
`different concentrations ofplastic-bound antl-CD3 monoclonalantibody in the absence
`(empty symbols) orin the presence(filled symbols) of anti-CD28.b, IL-12 p70production
`Figure 3 Rapid production of IFN-y following stimulation of CCR7~ memoryTcalls.
`by dendritic cells cultured with naive T cells (squares) orCCR7* memoryT calls (angles).
`CD45RA"CCR7* (a) and CD45RACCR7~ (b) CD4Tcells were stimulated for 7h with
`Dendritic cells were pulsed with toxic shack syndrometoxin (TSST) at 100 ng mI"(emply
`autologousdendritic cells pulsed with 100 ng mI" TSSTandstained with antibodies to
`symbols) or 1 ng mi"’ (filled symbols). Both T-call populations contained similar
`CD69 and IFN-y. CD69* cells were less than 2% in unstimulated cultures.
`proportions of V@2* cells,
`710
`
`Miltenyi Ex. 1029 Page 7
`
`

`

` supernatantusing an input of memory cells giving comparable proliferative responses,
`
`(%) Three subsets of peripheral blood CDE¢" cells were sorted by expression ofCD46)
`
`their relative proportions are not affected by recent antigenic
`stimulation. In all cases, IFN-y was produced by only the CCR7~
`| memory cells. Comparable results were obtained by analysing the
`response to tetanus toxoid or hepatitis B surface antigen in five
`primed donors, showing that both memory subsetscontain clonally
`expanded antigen-specific T cells, but these differ in their effector
`capacity. Thepresenceofexpandedantigen-specific memory cells in
`both subsets at different times after antigenic stimulation indicates
`that homeostatic mechanisms may maintain cells
`in both
`compartments’*”””*,
`Whatis the relationship between naivecells and thecells in the
`two memory subsets? When peripheral blood naive T cells were
`polyclonally stimulated, all cells became CD45R0* after 10 days
`| (data not shown), but mostofthecells retained CCR7 expression,
`whereas only a few acquired the capacity to produce IL-4 or IFN~y
`(Fig. 6a—c). When the CCR7* and CCR7 cells were sorted and
`stimulated, IL-4, IL-5 and IFN-y were found to be exclusively
`
`8
`
`o&
`
`
`
`
`
`IFN-y(ngmr")"|
`
`
`Figure5 Proliferative responses to recall antigens can be detectedin both CCR7* and
`CCR7~ subsets but notin naive T cells, Proliferative response of CD45RA* CCR7*naive T
`cells (squares), CD45RA°CCR7™(triangles) and CD45RACCR7” memory T cells (circles)
`in responseto tetanus toxoid presented by autologous monocytes. Respondercells from
`the same individual weretested 10 years after vaccination (empty symbols) and two
`weeks after a booster(filled symbols). Inset showsIFN-~y productionin the 24-h culture
`
`
`Table 1 Surface molecules on peripheralbloodnaive and memory CD4*
`T-cell subsets
`produced by the CCR7cells (Fig. 6d). Thus, with respect to the
`parametersanalysed,it appears that the samefunctionalsubsets of
`CD45RA*
`CD45RA™
`CD45RAr
`memory T cells that are detected in vivo can be generated by
`CCRT"
`CCR?”
`ccR7-
`stimulation ofnaive cells in short-term culture.
`>99
`>99
`>99
`
`Whenperipheral blood CCR7* memoryTcells were sorted and
`<0.4
`2
`3
`stimulated under the same conditions,almostall the cells that were
`cD25
`(%)
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`4
`8
`HLA-DR
`(%)
`<0.1
`1
`12
`recovered after 10 days had lost CCR7 expression and had acquired
`opis
`(MFI)
`38
`75
`the capacity to produceeffector cytokines uponfurther stimulation
`CDita
`(MFI)
`90
`218
`(Fig. 6e—g), indicating that this subset is poised to generate effector
`CD11b
`(%)
`<0.1
`35
`cells. Finally, peripheral blood CCR7 memorycells, after stimula-
`cD29
`(MFI)
`0
`43
`tion and expansion,retained their CCR7” phenotype andeffector
`cD4sd
`(MFI)
`10
`33/2
`function (Fig. 6h—j). This indicatesthat,at least in vitro, there may
`CD49e
`(2%)
`<0.
`10
`bea stepwise differentiation from naiveTcells to CCR7* memory to
`CA
`(%)
`<0.1
`25
`cD103
`(%)
`<0.1
`1
`CCR7 memary/effector T cells. This possibility is supported by
`CXCR4
`(26)
`98
`4
`analysis ofthe telomere length, which decreases as a function ofcell
`CCR4
`(%)
`<0.1
`6
`division’’. As shown in Fig.6k, the length oftelomeresin peripheral
`CCR6
`(%)
`<0.1
`45
`CXCRS
`(%)
`<0.1
`61
`blood CD4subsets decreased progressively from naive to memory |
`CCR
`(%)
`<0
`14
`CCR3
`(%)
`.
`4
`CCRS
`
`e
`a
`h
`CD45RAt CCR7*+
`CD45RA- CCR7*||GD45RA- CCR7~
`and CCR7. The sorted cells were stained and analysed for the expression of adhesion moelcules
`| and chemokine receptors. MFI, mean fluorescence intensity.
`*Mean value of major and minor peak.
`
`letters to natureBd
`
`
`
`
`
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`3
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`1 6
`a é é é
`Figure6 Differentiation potential of naive and memory T-cell subsets, a—d, Loss of CCR7
`followingin vitrostimulation of naiveTcells correlates with acquisitionofeffector function.
`10,000
`CD4* naiveTcells (CD45RA*, CCR7*) weresorted from peripheral blood(a), stimulated
`1,000
`100,000
`with anti-CD3 + anti-CD28, expandedfor 10 daysin IL-2 andtested for their capacity to
`T cells per culture
`produce IFN-y and IL-4 (b) or for CCR7 expression(c). CCR7* (R1) and CCR7~ cells (R2)
`were sorted and immediately testedfor their capacity to producecytokines following
`Polyclonalstimulation (d). e—g, Rapid polarization of CCR7* memory T cells following in
`vitro stimulation, CD4*, CD45RA”, CCR7* T cells were sorted from peripheral blood (e),
`stimulated, expanded and tested for cytokine production (f) and CCR7 expression (g).
`h-j, CD4*, CD45RA”, CCR7~T cells Isolated and stimulated as above retained a stable
`effector phenotype. k, Length of telomeres in peripheral blood naive and memory CD4*
`T-cell subsets. kb, kilobases.
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` letters to nature—EE
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`Peripheral blood mononuclearcells were stained with a rat monoclonal antibody (mAb)
`17. Baron,J. L., Madr, J. A., Ruddle, N. H., Hashim, G. & Janeway, C. A. Jr Surfaceexpression ofalpha 4
`specific for CCR7 (3D12, IgG2a) followed bya fluorescein isothiocyanate (FITC)-labelled
`integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med, 177, 57-68
`(1993).
`mouse anti-rat IgG2a mAb (PharMingen)oralternatively by a phycoerythrin (PE)-
`18. Wu,L. et al, CCRS levels and expression pattern correlate with infectability by macrophage-tropic
`labelled goat anti-rat immunoglobulin polyclonal antiserum (Southern Biotechnology
`Associates). The 3D12 mAb completely inhibited migration ofperipheral bloodT cells in
`HIV-1, in vitro. J. Exp. Med, 185, 1681-1691(1997).
`19, Sallusto,
`F, Mackay, C. R. & Lanzavecchia, A. Selective expression ofthe eotaxin receptor CCR3 by
`response to secondary lymphoid tissue chemokine (SLC) and EBI1-ligand chemokine
`humanThelper 2 cells. Science 277, 2005-2007(1997).
`(ELC) (R.F, unpublished data) anddid notaffect the responseofT cells to mitogenic or
`20. Tang, H. L. & Cyster, J. G. Chemokine up-regulation and activated T cell attraction by maturing
`antigenic stimulation (ES., unpublished data). In addition, 3D12stained all cell lines that
`dendritic cells. Science 284, 819-822 (1999).
`expressed CCR7 messenger RNA, butdid not stain CCR7 mRNA-negative cells. In some
`21, Sallusto, F, Lenig, D., Mackay, C. R. & Lanzavecchia, A. Flexible programs ofchemokine receptor
`experiments, a mouse mAbspecific for CCR7 (10H5, IgG3; produced by L. Wu,
`expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187, 875-883 (1998).
`LeukoSite) was used with comparableresults. The following PE-, PC5- or APC-labelled
`22, Grewal, I. S. & Flavell, R. A. CD40 and CD154 in cell-mediated immunity. Annu, Rev. Immunol. 16,
`mouse mAbs were used in different combinations: anti-CD45RA (ALB11, IgG1); anti-
`111-135 (1998).
`CD45R0 (UCHLI,IgG2a); anti-CD3 (UCHTI, IgG1); anti-CD4 (13B8.2, IgG1); anti-
`23, Abbas, A. K., Murphy, K. M.& Sher, A. Functional diversity ofhelper T lymphocytes. Nature 383,
`CD68(B9.11, IgG1); anti-CD1 Ja (25.3, IgG1); anti-CD11b(Bearl, IgG1); anti-CD18(7E4,
`787-793(1996).
`IgG1); anti-CD49d (HP2/1, IgG1); anti-CD49e (SAM1,IgG2b);anti-CD29 (K20, IgG2a);
`24, Kagi, D,, Ledermann,B., Burki, K., Zinkernagel, R. M, & Hengartner, H. Molecular mechanisms of
`anti-CD103 (2G5, IgG2a); anti-CD69 (TP1.55, IgG2b); anti-CD25 (B1.49, IgG2a); anti
`lymphocyte-mediated cytatoxicity and their role in immunological protection and pathogenesisin
`HLA-DR(B8.12,IgG2b); anti-CD40L (TRAP-1, IgG1)(all from Immunotech); andanti-
`vivo, Annu. Rev. Immunol. 14, 207-232(1996).
`CLA (HECA-205, rat IgG1; PharMingen).Staining for chemokinereceptors wascarried
`25, Casamayor-Palleja, M., Khan, M. & MacLennan, I. C. A subset ofCD4+ memory T cells contains
`outusingthe following mouse mAbs (all producedat LeukoSite): anti-CCR1 (2D4, IgG1);
`preformed CD40 ligand thatis rapidly but transiently expressed on their surface after activation
`anti-CCR3 (7B11,IgG2a); anti-CCR4 (1G1,IgG1); anti-CCR5 (2D7, IgG2a); anti-CCR6
`through the T cell receptor complex. J. Exp. Med. 181, 1293-1301 (1995).
`(1LA9,IgG1); anti-CXCR3 (1C6, IgG);and anti-CXCR4(12G5, IgG2a). Cells were sorted
`26. Hamann,D,etal. Phenotypic andfunctional separationofmemoryandeffectorhumanCD8+Tcells.
`using a fluorescence-activated cell sorter (FACS Vantage) and analysed on a FACScalibur
`J. Exp. Med, 186, 1407-1418 (1997).
`(Becton Dickinson Systems).Sorted cellswere immobilized on poly-1-lysinecoated slides,
`27. Sprent, J., Tough, D. BF. & Sun, S, Factors controlling the turnover ofT memory cells. Immunol. Rev.
`fixed in 2% paraformaldehydeand permeabilized in 0.1% Triton-X100 beforeintracellular
`156, 79-85 (1997).
`staining with an anti-perforin mAb (264, IgG2b; PharMingen),followed by FITC-labelled
`28. Tanchot, C. & Rocha, B, The organization ofmature T-cell pools. fmmunol. Today 19, 575-579
`1998;
`goat anti-mouse immunoglobulin and propidium iodideto visualize the nuclei by
`confocal microscopy. Thelength oftelomeres was determined using a Teloquantkit
`29, siN. PB,Hathcock, K. S, & Hodes, R. J, Regulation oftelomerelengthandtelomerase inTandB
`(PharMingen).
`cells: a mechanism for maintaining replicative potential. Immunity 9, 151-157 (1998),
`
`Cytokine detection
`Acknowledgements
`Tcells were stimulated with 10 wg mI" anti-CD3 antibody (TR66, IgG1) and 10-7 M
`We thank K. Hannestadand K.Karjalainen for critical reading; J. C. Howardfor his help as.
`phorbol12-myristate 13-acetate (PMA;Sigma). Cytokine production was measuredin the
`
`‘wordsmith’;
`M. Dessing and A. Pickert forcell sorting; A. Hoyfor helpin the initial
`24-h culture supernatants by ELISA using matched pairs of antibodiesspecific for IL-2,
`experiments; LeukoSite Inc., Cambridge, Massachusetts for providing antibodies to
`IL-4, IL-5, IFN~y (PharMingen). For cytokinedetection atthesingle-cell level,T cells were
`chemokinereceptors; and L. Wu for providing the CCR7 and CCR4 antibodies before
`stimulated with 10” M PMA and 1 pgml"ionomycinfor 4h, or with autologous
`publication. The Basel Institute for Immunology was founded andis supported by
`dendritic cells pulsed with 100ng mI"TSST for 7h in 10 wg ml"brefeldin A. Cells were
`FHoffmann-La RocheLtd, Basel, Switzerland.
`fixed and permeabilized with PBS containing FCS (2%) and saponin (0.5%)and stained
`with FITC-labeled anti-IFN-y (IgG1) and PE-labelled anti-IL-4 (IgG2b) or PE-labelled
`| anti-CD69 mAbs.
`
`Correspondence should be addressed to ES.(e-mail: sallusto@bii.ch).
`
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