400000146001 itNA DEC 90
`STE ENBOCK ME,M,LA 4
`U N I V OF0141( 041AU WM LIBRAR
`550 dABCOCK DR
`MADISON (cid:9)
`
`mivewt4 T93606
`
``1310
`R
`
`Ex. 1041 - Page 1
`
`

`

`nature of nature
`
`18 May 1989
`Vol. 339 Issue no. 6221
`
`KEMAL AXON GUIDANCE
`
`A red-labelled axon coursing through
`a transplant (blue) in embryonic
`Xenopus brain. Rotation of portions of
`optic tract in the embryo deflects
`neurons growing from the retina
`towards the optic tectum, showing
`that growing axons are guided by
`local cues. See page 218.
`
`THIS WEEK
`
`Fusion doubts grow
`Workers from MIT's Plasma Fusion
`Center conclude that the y-ray
`spectral line observed in the
`Fleischmann—Pons cold-fusion
`experiment was "specious",
`page 183. See also News pages.
`Bees heading north
`African honey bees released in
`Brazil 30 years ago are now
`migrating through Central Mexico
`ata rate of 300 miles per year, so
`are expected in Texas by mid-
`1990. This threatens commercial
`bee-keeping as these bees are
`more difficult to manage than
`other types. New evidence
`suggests that the African bees
`are spreading as an essentially
`intact population, displacing the
`European gene pool rather than
`mating with European swarms.
`Pages 211, 213 and 181.
`A bright Idea
`The conversion of sunlight to
`laser light, the destruction of
`hazardous waste, and the pro-
`duction of high-tensile-strength
`fibres are among the possible
`applications for the high-flux
`solar concentrator, described on
`page 198. This device can con-
`centrate terrestrial sunlight by a
`factor of 56,000, producing an
`irradiance in excess of that of the
`solar surface.
`Enterotoxins and HLA
`A direct demonstration that
`staphylococcal enterotoxins bind
`with high affinity to HLA-DR, and
`must be intact to stimulate T
`cells, is presented on page 221.
`Antigen processing
`The fungal antibiotic brefeldin A,
`which blocks transport out of
`the endoplasmic reticulum,
`reversibly inhibits the presenta-
`tion of cytosolically-synthesized
`antigen in association with MHC
`class I molecules. Page 223.
`
`Vitamin deficiency
`Retinoic acid potentiates the
`production of growth hormone in
`response to thyroid hormone or
`glucocorticoids in vitro, suggest-
`ing that vitamin A deficiency may
`explain in part the lower growth
`rate of malnourished children,
`page 231.
`Zeolite catalysis
`Nuclear magnetic resonance
`reveals the fate of 29 organic
`species in the adsorbed phase
`during zeolite ZSM-5 catalysed
`conversion of methanol to hydro-
`
`carbons. The technique provides
`clues as to reaction pathways on
`molecular sieves and should
`assist the design of new shape-
`selective catalysts, page 200.
`Enzyme catalysis
`Altering specific residues in the
`active site of a serine protease
`unexpectedly broadens its sub-
`strate specificity. Structural
`studies show that this effect is
`probably due to increased flexi-
`bility of the active site 'specificity
`pocket', which in mutant proteins
`can accommodate substrates of
`various sizes. Page 191.
`Storing memories
`Long-term potentiation of synap-
`tic strengths has long been
`regarded as a possible mech-
`anism of storing information in
`the nervous system. Theoretical
`studies indicate that selective
`depression of synaptic efficacy
`is also required for efficient
`memory storage. Long-term de-
`pression is now demonstrated
`experimentally, pages 215 and
`175.
`Guide to Authors
`Vol. 339, page 76.
`
`Nature, (ISSN 0028-0836) is published weekly on Thursday. except the last week in December.
`by Macmillan Magazines Ltd (4 Little Essex Street, London WC2R 3LF). Annual subscription
`for USA and Canada US$275 (institutional/corporate), US$125 (individual making personal
`Dyment). USA and Canadian orders to: 'Nature, Subscription Dept, PO Box 1733, Riverton, NJ
`1188077-7333, USA. Other orders to Nature, Brunel Road. Basingstoke, Hants RG21 2XS, UK.
`Second class postage paid at New York. NY 10012 and additional mailing offices. Authorization
`to Photocopy material for internal or personal use, or internal or personal use of specific clients.
`1., I, granted by Nature to libraries and others registered with the Copyright Clearance Center
`(CCC) Transactional Reporting Service. provided the base fee of $1.00 a copy plus $0.10 a page
`I' Paid direct to CCC, 21 Congress Street. Salem, MA 01970, USA. Identification code for
`Nature: 0028-0836/89 $1.00 + $0.10. US Postmaster send address changes to: Nature, 65
`nleecker Street, New York, NY 10012. Published in Japan by Nature Japan K.K., Shin-Mitsuke
`Bldg, 36 Ichigaya Tamachi, Shinjuku-ku, Tokyo 162. Japan. ©1989 Macmillan Magazines Ltd.
`
`NATURE SAYS
`Congressional committees do not make good referees
`• Nuclear weapons negotiations are needed
`within NATO
`
`161-162
`
`NATURE REPORTS
`Baltimore hearing ends • Peer review considered •
`One UK research council? Irish research • Money for
`Landsat? • Computer standards row • More money for
`Australian science • Cold fusion abandoned in Japan •
`Movement on climatic change • Chinese student protests
`• Fire kills laboratory mice • Indian agency blasted • No
`163-169
`norms for HDTV (cid:9)
`
`CORRESPONDENCE
`Including: Defining entropy • Peer review of fusion? •
`Moral responsibility • Selfish academics (cid:9)
`
`COMMENTARY
`With dosimeter in the sarcophagus
`Yuri Kanin (cid:9)
`
`NEWS AND VIEWS
`Understanding hydrogen bonds
`John Maddox
`Zeolites: Shaping up for catalysis
`John Dwyer
`Memory: Must what goes up come down?
`Richard G M Morris & David J Willshaw
`Anthropology: A tale of three cities
`J S Jones
`Mid-ocean ridges: Anatomy of the magma reservoir
`Ken C Macdonald
`Epstein—Barr virus: Synthesis for a complex role
`Bill Sugden
`Ceramic microcoils sprung for action
`No new fusion under the Sun
`Bertil Wilner
`Quantum optics: Laser gain without inversion
`Peter Knight
`Evolution: Neotropical African bees
`Robert E Page Jr
`Daedalus: Dry drinking
`
`SCIENTIFIC CORRESPONDENCE
`Problems with the y-ray spectrum in the Fleischmann et al.
`experiments R D Petrasso, X Chen, K W Wenzel,
`R R Parker, C K Li & C Fiore
`Fusion in from the cold? R Seitz • Mossbauer cancer
`therapy doubts D J Brenner, C R Geard & E J Hall
`Surface-plasmon microscopy W Hickel, D Kamp &
`W Knoll
`
`170
`
`171
`
`173
`
`174
`
`175
`
`176
`
`178
`
`179
`179
`
`180
`
`181
`
`181
`182
`
`183
`
`185
`
`186
`
`BOOK REVIEWS
`James J Gibson and the Psychology of Perception
`by E S Reed Brian Rogers
`RNA Genetics E Domingo et al. eds Stephen C Inglis
`A Theory of Human and Primate Evolution by C P Groves
`Bernard Wood
`Is Science Necessary? Essays on Science & Scientists
`by M FPerutz Desmond King-Hele
`
`187
`188
`
`189
`
`190 ►
`
`Ex. 1041 - Page 2
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`LETTERS TO NATURE
`
`by the newly described ER degradation system29•30. We have
`recently demonstrated that whether or not a protein within the
`ER is degraded can be directly correlated with the state of
`assembly of that protein'' and an analogous process for antigen
`would result in the degradation of any peptide that is not stably
`bound to class I molecules. The peptide-MHC complex then
`leaves the ER and travels through the Golgi apparatus to the
`plasma membrane. This model provides multiple testable
`hypotheses about the cell biology of endogenous antigen pres-
`entation which we are currently examining. (cid:9) q
`
`Received 31 January; accepted 6 April 1989.
`
`1. Unanue E. R. A. Rev. Immun. 2, 395 (1984).
`2. Morrison, L. A., Lukacher, A. E., Braciale, V. L., Fan, D. P. & Braciale. T. J. .1 exp. Med. 163, 903-921
`(1986).
`3. Yewdell, J. W., Bennink, J. F. & Hosaka. Y. Science 239, 637-640 (1988).
`4. Shimonkevitz, R., Colon, S.. Kappler, J. W. & Grey, H. M. J. lmmun. 133, 2067-2074 (1984).
`5. Maryanski, J. L, Pala, P., Corrudin, G., Jordan, B. R. & Cerrotini, J. C. Nature 324, 578-579 (1986).
`6. Townsend, A. R. M. et al. Cell 44,959-968 (1986).
`7. Gotch, F., Rothbard, J., Howland. K.. Townsend, A. & McMichael, A. Nature 326, 881-882 (1987).
`8. Babbitt. B. P., Allen, P. M., Matsueda, G.. Haber, E. & Unanue. E. R. Nature 317, 359-361 (1985).
`9. Guillet..1.-G., Lai, M.-L, Barter. T. J., Smith, J. A. & Getter, M. L. Nature 324, 260-262 (1986).
`10. Buus. S., Sette, A.. Colon. S., Jenis, D. M. & Grey. H. M Cell 47, 1071-1077 (1986).
`
`11. Townsend, A. R. M., McMichael, A. J., Carter, N. P., Huddleston, J. A. & Brownlee. G. G. Cell 39,
`13-25 (1984).
`12. Gotch, F., McMichael, A., Smith, G. & Moss, B. J. exp. Med. 165, 408-416 (1987).
`13. Townsend, A. R. M., Gotch. F. M. & Davey, J. Cell 42, 457-467 (1985).
`14. Townsend, A. R. M., Bastin, J., Gould, K. & Brownlee, G. G. Nature 324, 575-577 (1986).
`15. Townsend, A. et al. J. exp. Med. 168, 1211-1224 (1988).
`16. Moore, M. W., Carbone, F. R. & Bevan, M. J. Cell 54, 777-785 (1988).
`17. Takatsuki, A. & Tamura, G. Agric. Biol. Chem. 49, 899-902 (1985).
`18. Misumi, Y. et al. J. Biol. Chem. 261, 11398-11403 (1986).
`19. Lippincott-Schwartz, J., Yuan, L C., Bonifacino, J. S. & Klausner, R. D. Cell 66, 801-813 (1989,
`20. Kornfeld, R. & Kornfeld, S. A. Rev. Biochem. 64, 631-664 (1985).
`21. Hogan, K. T. et al. .1 exp. Med 168, 725-736 (1988).
`22. Palese, P., Tobita, K., Ueda, M., & Cornpans, R. W. Virology 61, 397-410 (1974).
`23. Matlin. K. & Simons. K. Cell 34, 233-243 (1983).
`24. Owen. M. J., Kissonerghis, A.-M., & Lodish, H. F. J. biol. Chem. 255, 9678-9684 (1980).
`25. Samelson, L. E.. Germain, R. N. & Schwartz, R. H. Proc. natn Acad. Sci. USA. 80, 6972-6976 (1983
`26. Ashwell, J. D., Cunningham, R. E., Noguchi, P. D. & Hernandez, D. J. exp. Med. 165, 173-194 (1.987,
`27. Bonifacino, J. S., Suzuki, C. K., Lippincott-Schwartz. J., Weissman. A. M. & Klausner, R. D. J. Ce-',
`Biol. (in the press).
`28. Walter, P. & Lingappa, V. R. A. Rev. Cell Biol. 2, 499-516 (1986).
`29. Lippincott-Schwartz, J., Bonifacino, J. S., Yuan, L C. & Klausner, R. D. Cell 54, 209-220 (1g881
`30. Chen, C., Bonifacino. J. S., Yuan, L. C. & Klausner. R. D. J. Cell Biol. 107, 2149-2161 (1988),
`31. Cowan, E. P.. Jelachich, M. L., Coligan, J. E. & Biddison, W. E. Proc. natn. Acad. Sci. U.S.A.
`5014-5018 (1987).
`32. Samelson, L E., Harford, J. B. & Klausner, R. D. Cell 4.3, 223-231 (1985).
`33. Sussman, J. J. et at. Cell 52, 85-95 (1988).
`
`ACKNOWLEDGEMENTS. We wish to thank Jennifer Lippincott-Schwartz. Eric Long, and Jonathan
`Ashwell for discussions and Richard Turner for technical assistance. Anti-influenza Monociong
`antibodies were the gifts of Dr. R. Webster. Brefeldin A was the gift of the Sandoz Co.
`
`Isolation of CD4- CD8-
`mycobacteria-reactive T
`lymphocyte clones from
`rheumatoid arthritis
`synovial fluid
`Joseph Holoshitz*, Frits Koningtt, John E. Coligant,
`Jacqueline De Bruyn§ & Samuel Strober*
`
`* Department of Medicine, Stanford University,
`Stanford, California 94305, USA
`t Biological Resources Branch, National Institutes of Allergy and
`Infectious Diseases, Bethesda, Maryland 20892, USA
`§ Pasteur Institute of Brabant, Brussels, Belgium
`
`THE majority of peripheral T cells express a heterodimeric, a/13
`T-cell receptor, which recognizes specific antigenic peptides bound
`to self major histocompatibility complex (MHC) molecules, and
`either the CD4 or CD8 surface markers. An additional subset of
`T cells1'2, whose physiological function is unknown, express a
`distinct CD3-associated receptor composed of y and 6 chains'.
`This subset includes cells lacking both CD4 and CD8 surface
`markers, which may be involved in autoimmunity3-6. The recogni-
`tion specificity of the y/6 receptor is not well characterized and
`has been defined in only one case to date, a murine cell line which
`shows MHC-linked specificity'. In this report, we describe the
`isolation of CD4- CD8-, y/8 TCR bearing T cell clones from
`the synovial fluid of a rheumatoid arthritis patient. These T cell
`clones respond specifically to mycobacterial antigens without
`MHC restriction.
`Our reasons for cloning these cells in the presence of mycobac-
`terial antigens were twofold. First, synovial cells, although poor
`responders to mitogens such as phytohaemagglutinin (ref. 8) or
`anti-CD3 antibodies9, have been shown to respond vigorously
`to mycobacterial antigens"°. Second, the association between
`the immune response to mycobacterial antigens and HLA-DR4
`(ref. 11), a class II MHC antigen commonly associated with
`rheumatoid arthritis'', suggested that this immune response
`might be related to the pathology of the disease. The proliferative
`
`# Present address: Department of Immunohaematology and Blood Bank, Bldg 1, E3-Q, University
`Hospital, 2300 RC Leiden. The Netherlands.
`
`226 (cid:9)
`
`responses of fresh synovial cells obtained from a 44-year-old
`patient with early rheumatoid arthritis are shown in Table I.
`There was a significant response to the acetone-precipitable
`fraction of mycobacterium tuberculosis (AP-MT) by synovial
`fluid cells but not by cells from the peripheral blood.
`To enrich for AP-MT reactive T cells, a T-cell line was estab-
`lished from this patient by repeated stimulations of synovial
`fluid T cells with AP-MT in the presence of irradiated,
`autologous peripheral blood mononuclear cells. The resultant
`line, JBT1, had a mixed phenotype. Flow cytometric analysis
`showed that 73% of the cells were CD4+CD8- and 23% were
`CD4-CD8- (Table 1). In unselected synovial fluid the per-
`centage of double negative cells was 11% and in peripheral
`blood it was 6%. Thus, double negative T cells were found in
`a higher percentage in the synovial fluid, and proliferated follow-
`ing repeated stimulations with the mycobacterial antigen
`AP-MT.
`We cloned line JBT1 by the limiting dilution technique and
`propagated fourteen clones successfully. Of the five that have
`been studied in detail, four were found to be double negative,
`whereas the fifth was found to have the CD4+CD8- phenotype.
`Figure 1 shows the fluorescence profiles of the CD4± clone 1.1
`and a representative double negative clone designated 1.2. All
`clones expressed CD2 and CD3 surface markers as well as DR
`antigens and IL-2 receptors but the double negative clones
`expressed neither CD4 nor CD8 (Fig. 1). Both types of clones
`did not stain positively with the following monoclonal anti-
`bodies: anti-Leu 7 or anti-Leu 9 (NK markers), anti-Leu 8
`(suppressor inducer marker), anti-Leu 12 (B cell marker) (data
`not shown). Whereas the CD4+ clone stained positively with
`anti-Leu 1 (CD5), the double-negative clones showed either dull
`or negative staining with this monoclonal antibody (not shown).
`The surface expression of T-cell receptor (TCR) chains was
`examined by staining with monoclonal antibodies. These
`included WT31, specific for an a//3 TCR framework deter-
`minant"; Ti-y A, specific for TCR y-chain14• and two mono-
`clonal antibodies specific for TCR 8-chain, 8 TCS1 (ref. 15)
`and TCR 81 (ref. 16). Flow cytometric analysis (Fig. 2a) showed
`that clone 1.1, a CD4± clone, had an alfl TCR, whereas the
`four double negative clones expressed y/6 heterodimers, as
`indicated by positive staining with both Ti-y A and TCR81 and
`no staining with WT31 antibody (Fig. 2a). The, S TCS1 anti•
`body, which binds many y/ 6 bearing cells", did not stain any
`of our y/6 T cell clones (Fig. 2a), although it stained PEER
`cells (not shown). It is conceivable that our double-negative
`clones express a different V8 gene product.
`
`NATURE • VOL 339 • 18 MAY 1989
`
`Ex. 1041 - Page 3
`
`

`

`LETTERS TO NATURE
`
`irnmunoprecipitation analysis using anti-peptide antisera was
`carried out to confirm that the CD3-associated TCR complexes
`expressed by the 1.2, 1.3, and 1.6 T cell clones represent y/5
`TCR complexes (Fig. 2b). Both the anti-TCR-y and anti-TCR-8
`sera precipitated the CD3-associated TCR, indicating that this
`receptor is a y/5 TCR. Because both antisera precipitate the
`1/5 complex it is not possible (without results of further experi-
`ments which are in progress) to determine which of the observed
`bands is y or 5.
`
`The increase in the percentage of double-negative T cells
`following repeated restimulations of the synovial fluid T cells
`with the mycobacterial antigen AP-MT (Table 1) suggested that
`the double-negative cells proliferated in response to AP-MT.
`To test this possibility, we measured proliferation of different
`clones to AP-MT using the thymidine incorporation assay (Table
`2). Both clone 1.1 of the CD4±, a/p TCR phenotype, and the
`four double-negative y/8 TCR clones proliferated in response
`to AP-MT. This proliferation was antigen-specific as the y/8
`
`CD2
`
`CD3
`
`CD4
`
`CD8
`
`DR (cid:9)
`
`IL-2R
`
`.•••••to (cid:9)
`
`411
`
`1.1
`
`1.2
`
`Relative number of cells
`
`Fluorescence Intensity
`
`I FIG. 1 FCM analysis of the surface marker phenotype of two representative
`
`clones. Five out of the fourteen clones obtained were studied more exten-
`sively. One clone, 1.1, was found to be of the CD4+ phenotype and four
`dones4.2.4.3, 1.4 and 1.6, had the double-negative phenotype represented
`rere by clone 1.2. Fluorescence intensity (x axis) as a function of relative
`cell number (y axis) of test antibodies (solid line) superimposed on back-
`ground fluorescence intensity obtained with isotype matched control anti-
`bodies (broken line) are shown.
`METHODS. The JBT1 T cell line was cloned by the limiting dilution technique
`based on the method described by Ottenhof et .31.21, with slight modifications.
`Briefly, viable JBT1 cells purified by Flaill gradient were diluted to give 0.5
`cells per 0.1 ml and plated into flat-bottom 96-microtitre plates (Costar), at
`
`0.1 ml per well over a feeder cell mixture, which consisted of autologous
`irradiated EBV-transformed B cells (106 cells m1-1), peripheral blood mononu-
`clear cells from three random donors (106 cell m1-1) and AP-MT (10 µg m1-1).
`Twenty-one wells contained growing cultures out of 96 wells originally
`seeded. Of these, fourteen clones were successfully propagated by repeated
`weekly cycles of restimulation with the feeder/antigen mixture, followed by
`expansion in IL-2-containing medium, The phenotypic analysis was done
`7-10 days after restimulation. Indirect flow cytometric analysis included the
`following monoclonal antibodies at the first stage: OKT 11 (anti-CD2, Ortho
`Diagnostics), anti-Leu 4 (CD3), anti-Leu 3 (CD4), anti-Leu 2 (CD8), L243
`(anti-DR), anti IL-2 receptor (all from Becton-Dickinson). For second stage,
`FITC-conjugated goat anti-mouse IgG (Tago) was used.
`
`TABLE 1 Proliferative responses and surface phenotype of peripheral blood, synovial fluid mononuclear cells, and the JBT1 anti-AP-MT cell line
`
`Cells derived from
`
`Peripheral blood
`Synovial fluid
`JBT1 Line
`
`Proliferative response
`(stimulation index ± s.d.)
`
`AP-MT
`
`3.2 ± 0.1
`22.3 ± 0.5
`19.0 ± 0.2
`
`PHA
`
`63.7 ± 0.6
`25.3 ± 0.6
`39.5±0.4
`
`Surface phenotype of T cells (% positive cells)
`
`CD4+
`
`CD8+
`
`CD4-CD8-
`
`TCR a/0
`
`TCR y/8
`
`67
`49
`73
`
`17
`40
`0
`
`6
`11
`23
`
`69
`62
`70
`
`4
`11
`ND
`
`Mononuclear cells were separated from fresh heparinized synovial fluid and peripheral blood on Ficoll-Hypaque gradients, The JBT1 anti-AP-MT T cell line
`was established by incubating synovial fluid mononuclear cells (106 m1-1) with AP-MT (prepared as previously describee; 10 fLg m1-1) in RPM! 1640 medium
`supplemented with 10% pooled normal human serum in 24-well plates (Costar). After 5 days, the blasts were resuspended for 7 days in medium containing
`10U ml-1 recombinant human IL-2 (Cetus), and then restimulated for 3 days with AP-MT in the presence of irradiated (3,000 R) autologous peripheral blood
`mononuclear cells in IL-2-free medium. After eight cycles of restimulation followed by expansion in IL-2, proliferation and phenotype were analysed.
`Proliferative responses of peripheral blood and synovial fluid mononuclear cells were measured as describee, incubating with AP-MT (10 lig m1-1) for 5
`days or phytohaemagglutinin (Wellcome Diagnostics 1µg m1-1) for 3 days. Proliferative responses of the JBT1 line were measured by incubating 15 x103
`cells and 75 x 103 irradiated autologous peripheral blood mononuclear cells per well with or without AP-MT (1014 m1-1) or phytohaemagglutlnin
`!0.25 FA, m1-1) for 3 days. [3H]thymidine (1 µCI per well) was added to all cultures 18 h before cell collection. Results are expressed as stimulation index
`(ratio of mean c.p.m. with antigen or mitogen/mean c.p.m. without antigen or mitogen). Control values for cultures without antigen or mitogen were 465 ± 31
`at 3 days and 514 ± 177 at 5 days for peripheral blood; 911 ± 168 at 3 days and 346 ± 73 at 5 days for synovial fluid mononuclear cells; and 510 ± 64
`for cell line JBT1. To determine the phenotype of the cells, synovial fluid mononuclear cells and simultaneously drawn peripheral blood mononuclear cells
`were enriched for T cells using the E-rosette technique. Viable JBT1 cells were purified over Ficoll gradients. For two-colour analysis, the following directly
`conjugated antibodies were used: anti-CD4 (Leu 3-FITC), anti-CD8 (Leu 2-FITC), anti-CD3 (Leu 4-PE) (all from Becton-Dickinson) and directly conjugated
`isotype-matched control antibodies (provided by Dr L. Herzenberg). Cells were stained with each of these antibodies alone and with combinations of
`anti-CD3 +anti-CD4, anti-CD3 + anti-CD8 and anti-CD3 +anti-CD4 +anti-CD8. For indirect immunofluorescent stainings, cells were stained with the following
`antibodies: anti-a//3 TCR (W131, Becton-Dickinson), anti-TCR y-chain (Ti-' A, a generous gift of Dr T. Hercend14), or an isotype-matched control antibody,
`followed by FITC-conjugated goat anti-mouse IgG (Tago). Flow cytometric analysis was performed using a B-D single Argon ion laser (488 mm) FACS III
`equipped with filters for fluorescein and phycoerythrin, interfaced to a Digital Equipment Corporation microVAX computer, using the FAGS DESK software.
`ND, not determined.
`
`NATURE • VOL 339 • 18 MAY 1989 (cid:9)
`
`227
`
`Ex. 1041 - Page 4
`
`(cid:9)
`

`

`LETTERS TO NATURE
`
`clones did not proliferate in response to myelin basic protein
`(Table 2) and various other control antigens (not shown).
`Although AP-MT is a relatively crude antigen, it is not mitogenic
`as indicated by its failure to stimulate both BF1 ( y/ (5) and 8.1
`(a / 13) control T cells clones derived from the peripheral blood
`of a normal individual (Table 2). BF1 and 8.1 are representative
`of y/ 8 and a/p alloreactive clones, respectively, which recogn-
`ize class I MHC antigens16. We are currently analysing the fine
`antigenic specificity of the double-negative clones. One clone
`(1.6), proliferated in response to P64, a biochemically purified
`heat-shock protein of relative molecular mass 64,000 from
`mycobacterium bovis BCG (Table 2).
`
`The CD4± clone and the four double-negative clones differ
`in their MHC restriction pattern. As shown in Fig. 3, the CD44
`clone 1.1 proliferated only in the presence of autologous antigen.,
`presenting cells and its proliferation could be blocked by anti.
`DR antibodies (60% suppression). In contrast, the double nega.
`tive clones, represented in Fig. 3 by clone 1.3, proliferated in
`the presence of antigen-presenting cells obtained from different
`unrelated individuals. This proliferation could not be blocked
`by anti-class I or class II HLA antibodies. HLA class I-negative
`Daudi cells could present AP-MT to the double negative clones,
`whereas K562 cells, which do not express both class I and class
`II MHC antigens, could not. No proliferative response to AP-
`
`a (cid:9)
`
`WT31
`
`TI - T A
`
`8 TCS1
`
`TCREI1
`
`.-.•.. too
`
`.ro...
`
`1 .1
`
`t. .
`
`1.2 (cid:9)
`
`1.3
`
`II
`
`—98
`
`- 68
`
`-43
`
`- 28
`
`-18
`
`1
`
`anti-CD3—anti-d
`
`anti-CD3—anti-d
`
`0
`..4
`
`anti-CD3—anti-CD3
`
`9
`
`1.2
`
`1.3
`
`1.4
`
`1 . 6
`
`...
`
`:Iwo (cid:9)
`
`Ho
`
`\
`\...
`
`ift\
`
`'..\-...1
`
`I
`
`V'"\.
`, ,
`
`r
`
`t (cid:9) t.
`
`:rut. toot
`
`1
`
`.
`
`1
`i
`
`
`
`ts
`
`Relative number of cells
`
`....t, i
`
`4.1.1, Mt
`
`.•••,t. mi.
`
`Fluorescence intensity
`
`FIG. 2 Surface expression of TCR chains by the clones. a, The CD4+ clone
`1.1 and the four double-negative clones 1.2-1.6 were analysed by indirect
`immunofluorescence using the following antibodies: WT31 (Becton-Dick-
`inson), specific for a/p framework determinant; Ti- y A, specific for TCR
`y-chain14, S-TCS1 (T Cell Sciences), specific for TCR S-chain, and TCR 81
`(prepared by Dr M. Brenner22 and provided by Dr L. Lanier), specific for TCR
`8-chain. Fluorescence of test antibodies (solid line), superimposed on
`fluorescence intensity obtained with a control antibody (broken line) is shown.
`b, Immunoprecipitation analysis. Cells from double-negative clones 1.2, 1.3
`and 1.6 were surface-iodinated and lysed in digitonin lysis buffer as pre-
`viously described23. After preclearing, immunoprecipitations were carried
`out using an anti-human CD3 8 serum24 (provided by A. Weiss). The
`
`228
`
`precipitates were washed and TCR-CD3 complexes eluted by incubating for
`10 min at 80 °C in 200 pi of a 1% SDS solution. After centrifuging the PAA
`beads, the supernatant was removed and divided in four equal aliquots. One
`of these was directly analysed on SDS-PAGE under reducing conditions (lane
`1). NP40 (200 p.1, 1.5%) was added to the other aliquots and these were
`subsequently used for reprecipitation with anti-CD3-8 serum (lane 2), anti-
`CD3- y serum (lane 3) or with anti-TCR-S serum (lane 4) before analysis on
`SDS-PAGE under reducing conditions. Molecular weight markers are indicated
`on the right. The anti-human CD3-8 and TCR y-chain sera were made against
`peptides corresponding to the C-terminal 14 and 7 residues of these chains
`respectively2325.
`
`NATURE VOL 339 • 18 MAY 1989
`
`Ex. 1041 - Page 5
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`LETTERS TO NATURE
`
`TABLE 2 CD4+ and double negative clones proliferate in response to AP-MT
`
`cell clone
`
`1.1
`1.2
`1.3
`1.4
`1.6
`BF1
`8.1
`
`Phenotype
`
`CD4+, a/13
`DN, y/8
`DN, y/B
`DN, y/8
`DN, y/6
`DN, y/8
`CD8+, a//3
`
`AP-MT
`
`12.3+0.1
`9.4+0.1
`12.0+0.2
`4.1 + 0.1
`7.4+0.1
`1.5+0.1
`1.3 + 0.2
`
`Proliferative response (stimulation index + s.d.)
`MBP
`P64
`
`ND
`2.6 + 0.1
`1.210.1
`1.0+0.0
`0.9 + 0.2
`ND
`ND
`
`ND
`ND
`ND
`ND
`5.4 + 0.1
`ND
`ND
`
`PHA
`
`15.8 + 0.1
`39.4 +1.1
`33.6+ 0.4
`7.5+0.1
`11.6+ 0.2
`16.9+0.3
`37.7 ±0.5
`
`proliferation assays were carried out as described in the legend to Fig. 1 using 15 x103 T cells and 75 x103 irradiated (3,000 R) autologous peripheral
`d mononuclear cells per well in the presence or absence of AP-MT (10 .cg m1-1) guinea pig myelin basic protein (MBP, 2014 m1-1, provided by Dr L.
`ieinman), a biochemically purified 64 K heat shock protein of mycobacterium bovis BCG (P64, 11.4 m1-1, prepared as described26) or phytohaemagglutinln
`ffiA; 0.25 pg m1-1). Cultures were pulsed with [3H] thymidine at 54 h and harvested at 72 h. A representative experiment, one of at least five repetitions
`g shown for each clone. Mean c.p.m.± s.d. obtained without antigen were 1,238 ± 242, 364+142, 399+57, 1,513+437 and 624+115 for clones 1.1,
`a 1.3, 1.4, and 1.6 respectively. Control clones of the double-negative, y/8 (BF1) or CD8+ a/p (8.1) phenotype were derived from the peripheral blood
`a normal individual as described16 and were provided by Drs A. Rivas and E. Engleman. Their mean c.p.m. + s.d. without antigen were 138+ 7 and
`oit 120 respectively. ND, not determined.
`
`ANTIGEN.
`PRESENTING
`CELLS
`
`ANTIBODY
`ADDED
`
`AUTOLOGOUS
`
`ANTI HLA ABC
`DP
`Do
`DR
`
`ALLOGENEIC 1
`2
`3
`4
`5
`6
`7
`8
`
`DAUDI
`K562
`XENOGENEIC
`NONE
`
`1.3
`
`illy
`
`• (cid:9)
`
`MEI
`
`ND
`
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`
`0 100 200 0 100
`RELATIVE PROLIFERATION MI
`
`FIG. 3 Proliferation of double-negative clones to AP-MT is MHC non-
`restricted. The double-negative clones, represented here by clone 1.3 (left
`side) proliferated in the presence of both autologous and allogeneic antigen-
`presenting cells obtained from randomly selected nonrelated individuals.
`HLA typing was disaparate among the allogeneic and autologous cells (not
`shown). Proliferation of the double-negative clones to AP-MT could not be
`blocked by anti-class I or class II antibodies.
`METHODS. Proliferation assays were as described in the legend to Table 2.
`Autologous or allogeneic peripheral blood mononuclear cells, or xenogeneic
`(Lewis rat spleen) cells, or Daudi and K562 cells were used as a source of
`irradiated antigen-presenting cells. Experiments were done in the presence
`and absence of these antigen-presenting cells, and in the presence or
`absence of AP-MT (10 lig m1-1). Anti-HLA monoclonal antibodies were added
`as described26. These included anti-HLA ABC (W6/32, Accurate), anti-HLA
`DP (37/21, B.D.), anti-HLA DQ (Leu 10, B.D.), or anti-HLA DR (L 243, B.D.).
`Results are presented as relative proliferative response (stimulation index
`obtained with AP-MT in the specified test conditions divided by the stimula-
`tion index obtained with AP-MT in the presence of autologous antigen-
`presenting cells, x100).
`
`MT could be found in the absence of antigen-presenting cells,
`or when xenogeneic cells were used (Fig. 3). Taken together,
`these results indicate that recognition of AP-MT by the double-
`negative clones was not restricted by MHC and did not require
`Interaction with class I MHC antigens. Recognition may,
`however, require presentation by a nonpolymorphic determinant
`of class II MHC gene product, or by different cell-surface
`antigens not expressed on K562 cells. This issue may be
`addressed by the use of cell lines transfected with class II MHC
`encoding genes.
`cells bearing the double negative phenotype have been
`replicated in a variety of murine autoimmune conditions3-16.
`For example, L3T4-, Lyt-2- T cells were shown to be capable
`
`of helping B cells to produce IgG antibodies to DNA in three
`systemic lupus erythematosus mouse strains's. Moreover, double-
`negative splenic T cells from normal mice were found to be
`capable of breaking oral tolerance" and to serve as precursors
`of autoreactive T cells6. Non-malignant, human, double nega-
`tive, TCR y/8-bearing cells have been isolated from the
`peripheral blood of a normal adult", a fetus' and an
`immunodeficient patient', but as yet there is no evidence to
`implicate them in the pathogenesis of autoimmune diseases.
`Our data show that double-negative y/6 cells can proliferate
`in response to a specific soluble antigen. The clones we describe
`were isolated from an autoimmune inflammatory site, and as
`such may be pathogenically relevant. (cid:9)
`0
`
`.eived 21 February: accepted 21 March 1989
`
`Brenner. M. B. of at Nature 322, 145-149 (1986).
`Lanier. L. & Weiss, A. Nature 324, 268-270 (1986).
`3 Morse, H. C. et al. J. lmmun. 129, 2612-2615 (1982).
`4 Hashimoto, Y., Maxam, A. M. & Greene, M. I. Proc. Nato Acad Sci. U.S.A. 83, 7865-7869 (1986).
`5 Delta, S. K., Patel, H. & Berry, D. J. exp. Med. 165, 1252-1268 (1987).
`6 Morisett, J. et al. Eur. 1. Immure 18, 387-394 (1988).
`7 Malls, L. A., Cron, R. & Bluestone, J. A. Nature 330, 262-264 (1987).
`8 Abrahamsen, T. G.. Froland, S. S. & Natvig, J. B. Scared 1. lmmun. 7, 81-90 (1978).
`8 lotz, M. et al. J. clin. Invest. 78, 713-721 (1986).
`r. Holoshitz, J. et al. Lancet II, 305-309 (1986).
`:1 Ottenhoff, T. H. M. et at Lancet II, 310-313 (1986).
`:1 Stansy, P. New Engi. 1. Med. 298, 869-871 (1978).
`:3 Sails, H. et al. J. lmmun. 135, 1922-1928 11985).
`litsukawa, S., Faure, F.. Lipinski, M., Triebel, F. & Hercend, T. J. exp. Med 166, 1192-1197 (1987).
`Hung, P. Nature 330, 501-502 (1987).
`2 Rivas. A., Koide, J., Cleary, M. L. & Engleman. E. G. J. Immun. 142, 1840-1846 (1989).
`
`17. Kitamura, K. et al. J. lmmun. 139, 3251-3259 (1987).
`18. Ferrini. S. et at J exp. Med 166, 277-282 (1987).
`19. Moingeon, P. et at Nature 323, 638-640 119861.
`20. DeBruyn, J. et at. Infect. lmmun. 55, 245-252 (1987).
`21. Ottenhoff, T. H. M. et al. Nature 329, 66-68 (1986).
`22. Band, H. et at Science 238, 682-684 (1987).
`23. Koning. F. et al. J. exp. Med 166, 595-600 (1987).
`24. Loh. E. Y. et at Nature 330, 569-572 (1987).
`25. Lew. A. M. et al. J. lmmun. 138, 807-814 (1987).
`26. Ottenhoff, T. H. M., Elf erink, D. G., Termljtelan, A., Koning, F. & de Vries, R R. P. Human lmmun.
`13, 117-123 (1.985).
`
`ACKNOWLEDGEMENTS. We thank Drs T. Hercend, L. Lanier. L Herzenberg, and A. Weiss for antibodies
`and Drs A. Rivas and E. Engleman for BF1 and 8.1 cells. We also thank S. Jones and B. Holm for
`technical assistance and K. Oto for secretarial help. This work was supported by research grants
`from the National Institutes of Health and the Northern California Chapter of the Arthritis Foundation.
`Dr I Holoshitz was supported by Rothschild F