|SoN 0009-9104
`
`Volume 127 Number 1 January 2002
`
`toCliniest and
`Experimental
`Emmunology
`
`
`
`
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`EDITOR-IN-CHIEF: A. P. WEETMAN
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`ASSOCIATE EDITORS:
`D. M. ALTMANN, B. P. MORGAN,
`D. B. G. OLIVEIRA, S. L. ROWLAND-JONES,
`A. D. B. WEBSTER & D. C. WRAITH
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`241011/31
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`CLINICAL AND EXPERIMENTAL IMMUNOLOGY
`EDITOR-IN-CHIEF: A. P. Weetman
`ASSOCIATE EDIToRS: D. M. Altmann,B. P. Morgan,D.B. G. Oliveira, S. L. Rowland-Jones,
`A. D. B. Webster & D. C. Wraith
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`AQELIBRARY.Qe
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`ABS‘i
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`Miltenyi Ex. 1020 Page 2
`ed#w#seseseseeeearr
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`Miltenyi Ex. 1020 Page 2
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`

`

`Clinical and
`Experimental
`Immunology
`
`CONTENTS
`
`Editorial review
`
`J. S. H. Gaston: Heat shock proteins and innate immunity
`1
`Review
`
`4 R.J. Boyton & D. M. Altmann: Transgenic models of
`autoimmunedisease
`
`Animal studies
`“12 LM. Bagenstose, R. Class, P. Salgame & M. Monestier:
`B7-1 and B7-2 co-stimulatory moleculars are required for
`mercury-induced autoimmunity
`20 J. Riemensberger, A. Bohle & S. Brandau: IFN-gamma
`and IL-12 but not IL-10 are required for local tumour
`surveillance in a syngeneic model of orthotopic bladder
`cancer
`
`27 CL. Montes, E. I. Zuftiga, J. Vazquez, C. Arce & A.
`Gruppi: Trypanosomacruzi mitochondrial malate
`dehyrogenasetriggers polyclonal B-cell activation
`37 A. S. Hansson, S. Lu & R. Holmdahl: Extra-articular
`cartilage affected in collagen-induced, but not pristane-
`induced,arthritis models
`
`43 L. A. Chau, S. Rohekar, J. J. Wang, D. Lian,S.
`Chakrabarti, L. Zhang, R. Zhong & J. Madrenas: Thymic
`re-entry of mature activated T cells and increased
`negative selection in vascularized allograft recipients
`53S. Perrier, B. Kherratia, C. Deschaumes, S. Ughetto, J. L.
`Kemeny, M. Baudet-Pommel & B. Sauvezie: 1L-1ra and
`IL-1 production in humanoral mucosalepithelial cells in
`culture: differential modulation by TGF-B1 and IL-4
`Basic immunology
`60 M. Cavers, B. A. Khoshkbijari, M. Macey, D. A.
`McCarthy, S. Irshad & K. A. Brown: Differential
`expression of $1 and {2 integrins and L-selectin on CD4*
`and CD8* T lymphocytes in human blood: comparative
`analysis betweenisolatedcells, whole blood samples and
`cryopreserved preparations
`
`D
`
`Blackwell
`Publishing
`
`IIMMA
`
`0009-9104 (200201)127:01;1-P
`
`
`
`
`
`
`
`Volume 127 Number 1 January 2002
`
`, British Society for
`
`immunologg
`
`Miltenyi Ex. 1020 Page 3
`
`66
`
`T. H. J. MacDougall, R. J. Shattock, C. Madsen, B. M.
`Chain & D.R. Katz: Regulation of primary HIV-1isolate
`replication in dendritic cells
`
`72K. Ueki, K. Tabeta, H. Yoshie & K. Yamazaki: Self-heat
`shock protein 60 induces tumournecrosis factor-a in
`monocyte-derived macrophage:possible role in chronic
`inflammatory periodontal disease
`
`Clinical studies
`
`78 M. Tarkowski, S. Chrul & J. Bodalski: The effect of IL-18
`and IL-12 induced CD30 expression and IL-4 and IFN-y
`production by allergen and PPD specific T cells
`
`85 S.J. C. Golby, C. Chinyama & J. Spencer: Proliferation
`of T-cell subsets that contact tumourcells in colorectal
`cancer
`
`92
`
`T. Akamizu, S. Ozaki, H. Hiratani, H Uesugi, J. Sobajima,
`¥. Hataya, N. Kanamoto, M. Saijo, Y. Hattori, K.
`Moriyama, K. Ohmori & K. Nakao: Drug-induced
`neutropenia associated with anti-neutrophil cytoplasmic
`antibodies (ANCA): possible involvement of complement
`in granulocyte cytoxicity
`
`99 R. Njemini, I. Meyers, C. Demanet, J. Smitz, M. Sosso &
`T. Mets: The prevalence of autoantibodies in an elderly
`sub-Saharan African population
`
`107 M. Sandmand, H. Bruunsgaard, K. Kemp, K. Andersen-
`Ranberg, A. N. Pedersen, P. Skinhgj & B. K. Pedersen: Is
`ageing associated with a shift in the balance between Type
`1 and Type 2 cytokines in humans?
`
`115 P.M. J. Kalkman, W. Fokkens, H. J. de Wit, J. P. van de
`Merwe, H. Hooijkaas, J. M. W. van Haarst, H. C.
`Hoogsteden & H. A. Drexhage: A hampered
`chemottractant-induced cytoskeletal rearrangementin
`granulocytes of patients with unexplained severe chronic
`and relasping infections of the upper and lowerairways.
`In vitro restoration by G-CSF exposure
`
`Contents continued on inside back cover
`
`Information on this journal can be accessed at
`http://www.blackwell-science.com/cei/
`
`This journal is available online at Blackwell Synergy. Visit
`www.blackwell-synergy.com to search thearticles and register for table
`of contents e-mail alerts.
`
`synergy 69
`
`Miltenyi Ex. 1020 Page 3
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`
`
`cml
`
`
`
`weeses
`
`
`
`AEEARTY“TINCWI
`
`wowfeaCe £&
`
`SUMMARY
`Flow cytometric analysis was used to compare the expression of adhesion molecules on human cD4t
`and CD8* T lymphocytes in isolated blood mononuclear cells (MNCs) in whole blood samples and in
`cryopreserved MNC preparations. Examination of MNCs revealed that
`the CD11b and CD11c
`components of the 82 integrins were preferentially expressed on CD8*Tcells, whereas CD62L was
`present on more CD4* T cells. All CD4* and CD8* T lymphocytes were positive for CD11a but the
`CD8* population had a higher intensity of expression of CD11a and also CD11b. Virtually identical
`results were obtained with T cells in whole blood samples. In relation to the 61 integrins, the only
`difference between isolated CD4* and CD8* T cells was thatthe latter subset had a greater proportion
`of cells bearing CD49d. The naive cell marker CD45RA was present on the majority of CD8*Tcelis
`whereas CD45RA and the memory marker CD45RO were evenly distributed within the CD4*Tceil
`subset. Although cryopreservation of lymphocytes did not modify the expression of 81 and 82 integrins
`it produced a marked reduction in the percentage of CD4* and CD8* T cells bearing CD62L. With
`regard to endothelial interactions, it appears that cryopreserved lymphocytesare suitable for inclusion in
`studies of integrin-mediated adhesion butnot for those relating to tethering or recognition of addressins
`on high endothelial venules. Differences in adhesion molecule expression between CD4* and CD8* T
`lymphocytes could underlie the selective extravasation of these subsets into sites of infection and
`inflammation.
`
`
`
`Clin Exp Immunol 2002; 127:60-65
`
`Differential expression of 81 and £2 integrins and L-selectin on CD4* and CD8*
`T lymphocytes in human blood: comparative analysis betweenisolatedcells,
`whole blood samples and cryopreserved preparations
`
`M. CAVERS,B. AFZALI KHOSHKBIJARI, M. MACEY*,D. A. MCCARTHY*,S. IRSHAD & K. A. BROWN
`Departmentof Immunobiology, The Guy’s, King's College and St Thomas’s Hospitals’ Medical and Dental School (GKT,) and
`*Department of Haematology, The Royal London Hospital, London UK
`
`(Accepted for publication 4 September 2001)
`
`Keywords
`
`adhesion molecules
`
`flow cytometry
`
`human T lymphocytes
`
`INTRODUCTION
`
`Surface adhesion molecules on T lymphocytes control extravasa-
`tion into lymphoid and non-lymphoid tissue [1,2]. Lymphocyte
`attachment to blood vessel walls is governed in part by the 61
`integrins (CD49/CD29 family)
`recognizing vascular adhesion
`molecule-1 (VCAM-1) and componentsof the extracellular matrix
`and by membersof the 82 integrins (CD11/CD18 family) binding
`to intercellular adhesion molecule-1 (ICAM-1). The initial
`tethering of T lymphocytes to endothelial cells, which precedes
`the firm adhesion mediated bythe integrins, is dependent upon the
`function of CD62L [2], which is a receptor that interacts with
`addressins on high endothelial venules and is responsible for the
`‘homing’ to secondary lymphoidtissue of circulating lymphocytes
`[3]. Adhesion molecules also facilitate the engagement of T
`lymphocytes with antigen-presenting cells. This binding allows
`
`Correspondence: Dr K A Brown, Department of Immunobiology, 3rd
`Floor New Guy’s House, Guy’s Hospital, London SE1 9RT, UK.
`E-mail: alun.brown @kcl.ac.uk
`
`60
`
`the T cell receptor to scan the surface of the antigen-presenting
`cell for antigen and to sustain cell contact, should antigen
`recognition occur,
`in order to initiate and complete effector
`mechanisms.
`Quantitative or qualitative variations in surface adhesion
`molecules are likely to contribute to the selective extravasation of
`distinct classes of leucocytes. Most lymphocytes express CD49d/
`CD29 and very few CD11b,in contrast to neutrophils, which are
`virtually all positive for CD11b but negative for CD49d [4,5].
`Differences in adhesion molecule expression may be responsible
`for the preferential sequestration of CD4* T cells and CD8* T
`cells in lymph nodes and Peyer’s patches, respectively [6], to the
`selective migration of CD4* T cells
`to sites of delayed
`hypersensitivity and chronic inflammatory lesions [7] and to the
`distinct
`functional
`roles of CD4* and CD8* T cells. To
`investigate whether these subsets exhibit characteristic adhesion
`molecule profiles, we compared the distribution and level of
`expression of CD1la, CD11b, CDllc, CD49d, CD29 and L-
`selectin on circulating human peripheral blood CD4* and CD8* T
`© 2002 Blackwell Science
`
`Miltenyi Ex. 1020 Page 4
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`TALLWESELTGS
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`
`Differential expression of B1 and B2 integrins and L-selectins
`
`61
`
`cells in whole blood samples and in mononuclear leucocytes
`jsolated from normal healthy subjects. The phenotypic and
`functional assessment of lymphocytes from patients with various
`disorders are occasionally hampered by the late arrival of blood
`samples to the laboratory. Cryopreservation of the cells over-
`comes this problem and this procedure is also advantageous to
`lymphocyte studies in longitudinal investigations. Accordingly,
`another feature of the present study was to ascertain whether the
`process of cryopreservation and thawing modified the expression
`of adhesion molecules on CD4* and CD8* T lymphocytes.
`
`MATERIALS AND METHODS
`
`Antibodies and reagents
`Hanks’s Balanced Salt Solution (HBSS, without phenol red),
`HEPES buffer, bovine serum albumin (BSA), fetal bovine serum
`(FBS), dimethyl sulphoxide (DMSO) and Histopaque-1077 were
`all obtained from Sigma Chemical Co, Irvine, UK. Fluorescein
`isothiocyanate
`(FITC)-conjugated antibodies
`against CDlla
`(25-3), CD11b (Bear-1), CD29 (K20), CD49d (HP2/1), CD62L
`(DREGS56), CD4 (13B8-2), CD8 (B9-11), phycoerythrin-cyanine
`5.1
`(PC5)-conjugated antibody against CD3 (UCHT1) and
`phycoerythrin (PE)-conjugated antibodies against CD4 (13B8-2),
`CD8 (B9-11) and CD11c (BU15) were purchased from Immuno-
`tech (Marseille, France). Other FITC-conjugated antibodies to
`CD49b (AK7), CD49e (SAM1), CD49f (450-30 A), CD45RA
`(F8-11-13) and CD45RO (UCHLI) were obtained from Serotec
`(UK). Isotype matched conjugated negative control antibodies
`(igG1, IgG2a and IgG2b) were purchased from each of the above
`manufacturers. Red cell
`lysing solution,
`‘FACSlyse’
`(Becton
`Dickinson) was diluted 1 in 10 (v/v) from stock with distilled
`water and the fluorescent nuclear dye LDS-751 (Molecular
`Probes, Leiden, the Netherlands) was diluted 1 in 100 (v/v) from
`a stock solution of 0-02% (v/v) in methanol using a 0.5% (v/v)
`solution of formaldehyde in HBSS as a diluent.
`
`Processing of blood samples
`Blood samples were obtained from 38 normal human volunteers.
`Experiments were performed on either fresh whole blood (six
`females,
`six males, age range 21-52 years) or on isolated
`mononuclear cells (17 females, nine males, age range 20-—
`63 years). For
`the whole blood analysis, 4 ml blood were
`collected in K, EDTA tubes (Becton-Dickinson). For analysis of
`isolated mononuclear cells (MNCs), 30 ml venous blood from
`each subject were collected in lithium heparin tubes (Becton-
`Dickinson), diluted 1:
`1
`(v/v) with warmed saline solution,
`layered onto Histopaque-1077 and centrifuged at 800 g for
`20 min. The MNC layer was washed twice in HBSS with 1%
`HEPES and aliquots prepared for immediate flow cytometric
`analysis or cryopreserved for later study.
`
`Labelling of lymphocytes with antibodies
`Whole blood samples. For each analysis 25 yl anticoagulated
`whole blood were labelled with 5 1 of antibody against either
`CD4 or CD8 and with 5 wl of an antibody against one of the
`adhesion molecules under
`investigation. Each sample was
`incubated for 10 min on ice in the dark followed by the
`addition of a further 75 wl HBSS. Red cells were lysed by
`adding 2 ml FACSlyse to each sample for 10 min in the dark at
`room temperature. Samples were centrifuged at 300 g for 5.min,
`the supernatant removed, the pellet resuspended in 1 ml HBSS
`and further centrifuged at 200 g for 5 min The supernatant was
`aspirated and 0-5 ml LDS nuclear stain solution added to each
`sample. At the same time, positive and negative single antibody
`controls were set up with 5 1 of PE-conjugated antibody against
`CD4 and CD8, 5 1 of FITC-conjugated antibody against each of
`the adhesion molecules tested, or 5 xl of isotype-matched PE-
`and FITC-conjugated mouse IgG1 antibodies.
`
`Isolated cells. For each test, 2-5 x 10° MNCs in 200 pl
`labelling solution (see Cryopreservation of mononuclear cells)
`were labelled with 5 41 PC5-conjugated antibody against CD3,
`10 1 FITC- or PE-conjugated antibody against CD4 or CD8 and
`5 wl or 10 ysl (as appropriate) FITC- or PE-conjugated antibody
`against the adhesion molecules or CD45. At the same timetriple-
`negative controls of mouse IgG1 PE-IgG1 PC5- and either mouse
`IgG1, IgG2a or IgG2b FITC-conjugated antibodies were prepared.
`Single positive controls were comprised of the PCS5-conjugated
`antibody against CD3, the PE-conjugated antibody against CD8
`and FITC-conjugated antibodies to each adhesion molecule. A
`further negative control of unlabelled cells was also used for each
`experiment. Each of the above preparations was incubated for
`20 min in the dark at room temperature. The cells were then
`washed in 1 ml HEPES-buffered HBSSby centrifugation at 600 g
`for 5 min and resuspended in 400 yz] labelling solution.
`
`Flow cytometry
`Whole blood samples underwent two-colour analysis on a Becton
`Dickinson FACScan flow cytometer using the Consort 32 Lysis II
`program. Lymphocytes were identified by their position on a dot
`plot of light forward versus side scatter as well as side scatter
`versus LDS-751 staining intensity (monitored in the FL3
`channel). Three-colour analysis was undertaken on the isolated
`MNCsusing a Coulter EPICS MCL XL2 flow cytometer and
`gating the lymphocyte population in the plot of forward versus
`side scatter. For each sample, 5000 events were recorded. By
`including the antibody to CD3 (monitored in the FL4 channel) and
`gating on the CD8/CD3 double positive lymphocytes within the
`FL2 versus FLA plot,
`the CD8*CD3~ natural killer cell
`population, which may appear within the lymphocyte gate, was
`excluded from analysis. Adhesion molecule expression was
`recorded as percentage of lymphocytes and mean fluorescence
`intensity as monitored in the double-positive quadrant of the FL1
`versus FL2 plot gated for CD3* lymphocytes (see Fig. 1).
`
`Cryopreservation of mononuclearcells
`MNCs, 7 x 10°, were suspended in 1 ml 90% heatinactivated
`FBS with 10% DMSO andstored in an insulated container at
`~80°C for 24-48 h before introduction into liquid nitrogen. Cells
`Were thawed rapidly on the day of analysis, washed in 20 ml cold
`HEPES-buffered HBSS and resuspended in HBSS, 1% HEPES
`buffer, 0.5% BSA (labelling solution) at a concentration of
`1-25 x 10°/ml.
`
`Statistical analysis
`The data were normally distributed. Hence, comparisons between
`CD4 and CD8 lymphocyte expression, as well as between
`cryopreserved and non-cryopreserved cells, were performed using
`Student’s paired t-test. P-values < 0-05 were set as the level of
`significance.
`© 2002 Blackwell Science Ltd, Clinical and Experimental Immunology, 127:60-65
`
`Miltenyi Ex. 1020 Page 5
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`62
`
`M. Caversetal.
`
`
`
`2
`
`“0°
`10°
`102
`10°
`10¢
`“10°
`10’
`10?
`10° 10°
`CD62L
`CD62L
`Fig. 1. Flow cytometric profile of CD3*CD4* and CD3*cpDst
`lymphocytes from an isolated population of MNCs, expressing the
`adhesion molecule CD62L.
`
`RESULTS
`
`Distribution of integrins and CD62L on CD4* and CD8* T
`lymphocytes in whole blood samples and isolated mononuclear
`cells
`The expression of CDlla, CDi1lb, CD1Ilc and CD62L was
`examined on CD4* and CD8* T lymphocytes in whole blood
`samples obtained from 14 healthy subjects. Figure 2a shows that
`
`P<0-001
`
`°
`
`; C
`
`D11a
`
`CD11c
`CD11b
`Adhesion molecules
`
`CD62L
`
`(b)
`
`100
`
`P<0-002
`
`CD11b was expressed on a higher percentage of CD8* T cells
`(mean 18 + 14%) than CD4* T cells (mean 2 + 2%; P < 0-001)
`and that CD11c waspreferentially associated with CD8*T cells
`(mean 7 + 8% versus mean 2 + 2% for CD4* T cells;
`P < 0-005). In contrast, the distribution of lymphocytes bearing
`CD62L was greater in the CD4* subset (mean 60 + 14%) than in
`the CD8* population (mean 44 + 18%; P <0-001). Virtually all
`CD4t and CD8* T cells displayed CDila on their surface with
`the CD8* subset (Table 1) exhibiting the higher intensity of
`expression of
`the molecules
`(mean MFI
`105 + 41 versus
`75 + 17; P <0-05). Of the other adhesion molecules, Table 1
`further showsthat relative to CD4* cells, the CD8* T cells had a
`higher MFI for CDi1b (P < 0-01) and a similar expression of
`CD1lic and CD62L. The results show that differences in the
`distribution of CD11b, CD11¢ and CD62L on CD4* and CD8* T
`cells in whole blood were retained upon isolation of the MNCs.
`For each adhesion molecule investigated the level of distribution
`(i.e. % stained cells) and MFI were remarkably similar on cbD4*
`and CD8* T cells in whole blood samples and in isolated MNC
`preparations.
`Since adhesion molecules may be up-regulated by isolation
`[8], the studies were extended to the measurement of the above
`adhesion molecules on CD4* and CD8* T lymphocytes in whole
`blood samples from an additional 12 subjects. In comparison with
`the CD4* population there were more CD8* T cells expressing
`P<0-001
`(a)
`CD11b (mean 25 + 16% versus 4 + 7%; P <0-001) and CD11c
`100;sees wme-
`(mean 8 + 8% versus 3 + 1%; P <0-005) but fewer cells
`—
`6
`bearing CD62L (mean 56 + 14% versus 75 + 12%; P <0-005).
`x 80
`Although CD11a waspresent onall lymphocytes, in comparison
`¢
`eo
`8
`with CD4* T cells the mean fluorescence intensity (MFI ofthis
`P<0005
`* *
`5 60
`integrin and CD11b was higher on CD8* T cells (P < 0-01).
`m1 o
`°
`There was no difference between CD4* and CD8*Tcells in their
`& 40
`~
`:
`MFI values for CD11c and CD62L.
`e
`3
`.o
`>
`ao
`:
`The next stage of the study assessed the expression of B!
`- 20
`integrins on CD4* and CD8* T cells in MNC preparations.
`Figure 2b shows that CD49d was moreprevalent on CD8*T cells
`(mean 60 + 18%)
`than CD4* T cells (mean 35 + 17%: P
`<0-002), but that CD29,
`the 6 chain of this subfamily, was
`equally distributed on CD8* T cells (mean 53%) and cpD4* T
`cells (mean 47%). Very few lymphocytes expressed CD49b,
`CD49e and CD49fandthe intensity of expression for all of the 61
`integrins was similar for both T cell subsets (Table 1).
`
`se 80
`w
`= 60
`o
`2
`a 40
`E
`=
`
`ce
`3
`&
`°
`to
`®
`§
`3s
`3?
`e
`
`:
`oe
`e :
`@ 2
`& e
`os
`°
`, 8
`
`0)
`
`CD49b
`
`CD49f
`CD49e
`CD49d
`Adhesion molecules
`
`CD29
`
`isolates CD8* T cells preferentially
`Fig. 2. Within mononuclear cell
`express CDi1b, CD1ic and CD49din relation to CD4* T cells but less
`CD62L. Blood mononuclear cells were isolated from 14 subjects and
`examined for their distribution of (a2) CD11a, CD11b, CD11c and CD62L
`and (b) CD49b, CD49d, CD49e, CD49f and CD29. Results are presented
`as the percentage of positively stained CD4* and CD8* T lymphocytes.
`Horizontal lines represent mean values. Differences (P-values) between
`CD4* and CD8* T cells in the distribution of adhesion molecules are
`indicated on the figure. O, CD4; @, CD8.
`
`Segeree
`
`Mertiefnew
`
`SOERELTWOO
`“TENAye
`AATATCEY
`vvfetataCBP”8
`
`
`
`
`
`
`
`Cryopreservation and the expression of adhesion molecules
`Mononuclear
`leucocytes were
`isolated from seven healthy
`subjects and aliquots of the cells were immediately analysed for
`their expression of CD1la, CD11b, CD11c, CD49d, CD29 and
`CD62L, whereas the remainder were cryopreserved. The stored
`cells were thawed approximately 1 month later and examined for
`their expression of the above adhesion molecules. Figure 3a
`showsthat with the fresh preparations of MNCs,the proportion of
`cells bearing CD11b, CD1 1c and CD49d washigher in the cpD4*
`T cell subset. Similar results were obtained with the cryopreserved
`MNCs(Fig. 3b). However, a majordistinction between the fresh
`and stored cells wasin their distribution of CD62L. With the non-
`cryopreserved cells (Fig. 3a), CD62L waspresent on the majority
`of CD4* T cells (mean 61 + 19%) in comparison to a mean
`40 + 22% (P < 0-002) of the CD8* T cells. This difference was
`not apparent within the cryopreserved cells (Fig. 3b) because
`CD62L was only expressed on a small minority (P < 0-001) of
`CD8* T cells (mean 12 + 15%) and CD4* T cells (mean
`
`Oe
`
`© 2002 Blackwell Science Ltd, Clinical and Experimental Immunology, 127:60-65
`
`Miltenyi Ex. 1020 Page 6
`
`Miltenyi Ex. 1020 Page 6
`
`

`

`
`
`Table 1. Intensity of expression of 82, 81 integrins and L-selectin on CD4* and CD87Tcells in isolated blood mononuclear leucocytes
`
`
`Differential expression of B1 and B2 integrins and L-selectins
`
`63
`
`MFI
`
`
`Number
`Number’
`of subjects
`cD4*
`cpD8*
`of subjects
`cD4*
`cps*
`
`
`B2 integrins
`
`#1 integrins
`
`CDila
`CD11b
`CD1ic
`CD62L
`
`75
`1542
`(5)
`CD49b
`2344
`25 +6
`(14)
`CD49d
`22 +21
`1t7
`(5)
`CD49e
`18 +3
`i541
`(5)
`CD49f
`3146
`3447
`(14)
`CD29
`et
`
`(14)
`(14)
`(14)
`(14)
`
`+17
`2243
`39 + 20
`35 + 13
`
`105 + 41*
`33 + 6**
`43 + 16
`37 + 14
`
`The intensity of expression of adhesion molecules is presented in terms of the mean fluorescence intensity (MFI). Results represent the mean values
`+ standard deviation of mononuclear leucocytes prepared from five or 14 healthy subjects. *P < 0-05, **P < 0-01 compared with expression on CD4* T
`cells.
`
`7
`
`Fig. 3. Adhesion molecule expression on CD4* and CD8* cryopreserved
`The intensity of expression of adhesion molecules is presented in terms
`T lymphocytes. The expression of CD11a, CD11b, CD11¢, CD49d, CD29
`and CD62L was measured on mononuclear cells isolated from the blood of
`of the mean fluorescence intensity (MFI). Results represent the mean
`values + standard deviation of mononuclear leucocyte preparations from
`seven healthy subjects
`(a) prior
`to cryopreservation and (b) after
`cryopreservation. Results are expressed as the mean percentage of cp4*
`seven healthy subjects. Lymphocytes were analysed for adhesion molecule
`expression immediately upon isolation (non-cryopreserved) or after
`and CD8* T lymphocytes bearing the adhesion molecules. Differences (P-
`approximately 1 month of cryopreservation. *P <0-05 compared with
`values) between CD4* and CD8*Tcells in the distribution of adhesion
`CD4* cells. **P < 0-02 compared with CD4* cells.
`molecules are indicated on the figure. 0, CD4; @, CD8.
`
`© 2002 Blackwell Science Ltd, Clinical and Experimental Immunology, 127:60-—65
`
`Miltenyi Ex. 1020 Page 7
`
`
`
`Examination offreshly isolated MNCsfrom five healthy subjects
`15 + 18%). From Table 2 it is apparent that the MFI of the
`revealed a trend for more of the CD8*Tcells to express CD45RA
`adhesion molecules investigated was not modified by cryopre-
`(mean 77 + 7%)
`than the CD4* T cells (mean 61 + 12%),
`servation and thawing.
`whereas approximately just under 50% of the CD4*Tcells and
`The last phase of the study investigated the distribution of
`CD8* T cells expressed CD45RO. Similar distributions in the
`CD45RO and CD45RA antigens on CD4* and CD8* T
`CD45RA and CD45RO subsets were apparent
`in the MNCs
`lymphocytes in isolated MNCsprior to and after cryopreservation.
`following cryopreservation except that the percentage of CD8*t T
`cells bearing CD45RA (mean 79 + 10%) was greater than the
`CD4* T cells (mean 65 + 9%; P <0-002).
`
`P<0-01
`
`P<0-002
`
`(a) 100
`20
`
`DISCUSSION
`
`that human blood T lymphocytes
`study demonstrates
`This
`expressing CD11b, CD11c and CD49d are more prevalent within
`the CD8* population than the CD4* population, although the
`CD8* subset contained fewer cells bearing CD62L. In addition,
`the CD8* T cells had a higher level of expression of CD11a and
`CD11b. Comparable results were obtained with preparations of
`
`Table 2. Intensity of expression of adhesion molecules on cryopreserved
`CD4* and CD8*T cells
`
`
`MFI
`
`
`Non-cryopreserved
`Cryopreserved
`
`
`Adhesion
`molecule
`cpD4*
`cps*
`cp4*
`cD8*
`
`
`125 + 48*
`69 + 21
`128 + 44*
`84 + 20
`CDila
`31 + 8**
`2343
`34 + 8**
`22 +3
`CD11b
`61 + 63
`87 + 86
`37 + 15
`26 + 10
`CD11c
`22 +3
`2345
`2444
`2647
`CD49d
`31+8
`33 +7
`3143
`3645
`CD29
`26 + 15
`35 + 12
`37 + 18
`38 + 17
`CD62L
`
`
`CD11a CDi1b CD1ic CD49d
`Adhesion molecules
`P<0-005
`
`+
`(b)
`
`CD29
`
`CD62L
`
`CDila CD11b CD11c CD49d
`Adhesion molecules
`
`CD29
`
`CD62L
`
`80
`
`60
`
`40
`
`20
`
`x2
`
`>
`re)
`$
`Q
`& 100
`EL
`80
`
`60
`
`40
`
`Miltenyi Ex. 1020 Page 7
`
`

`

`
`
`
`
`MIG
`
`WeeBwNehetdsZtou
`
`“etFfONTSere
`vyfreres4
`FOSAer
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`TINEE
`FRRRY
`™GFtpge
`
` 4
`
`|r
`
`*?
`i
`
`earlier observations [17]. Naive lymphocytes (CD45RA*), which
`leucocytes and whole blood samples.
`isolated mononuclear
`Differences between the expression of adhesion molecules on
`have yet to encounterantigen,recirculate from blood to secondary
`CD4* and CD8* T cells were maintained upon cryopreservation
`lymphoidtissue whereas circulating memory cells (CD45RO +),
`except for CD62L whosedistribution was significantly reduced in
`that have undergone antigen-induced clonal proliferation, prefer-
`both subsets.
`entially enter non-lymphoid tissue [24]. Such differences in
`Despite the known confinement of CD11b and CD1Ic to a
`migratory behaviour are associated in part with a greater
`proportion of the CD45RA* cells bearing CD62L, which is
`minority of T lymphocytes [9-11], we noted that in relation to
`CD4*T cells, more CD8* T cells had CD11b and CD11c ontheir
`compatible with the present finding, and with CD45RO™ cells
`surface and a higher expression of CD11b. The CD8* cells that
`generally possessing a higher expression of 81 and G2 integrins
`than CD45RA™cells[1,24]. Most of the LFA-1 on naiveT cellsis
`bear CD11b suppress T cell proliferation [12] and CD11c, which
`is expressed by somecytotoxic T cell clones, enhances cytolysis at
`considered to be in an inactive form, whereas memory cells
`the level of conjugate formation between effector andtargetcells
`predominantly express active molecules [25]. On this basis we
`would have anticipated the CD8*Tcells possess a lower surface
`[13]. The binding of cytotoxic T cells to epithelial cells and
`expression of integrins compared with CD4* T cells rather than
`fibroblasts [5,14] is augmented by CD11a, which also promotes
`lymphocyte attachment to endothelium by recognition of ICAM-1
`the converse, which wasa greater distribution of CD11b, CD11c
`and ICAM-2 [2]. CD11a waspresent on all CD4* and CD8* T
`and CD49d and a higher expression of CDlla and CD11b.
`lymphocytes and its level of expression was greater on CD8* T
`Moreover, our recent work reveals that an antibody directed
`against
`the active form of CD1la stains CD8*t T cells in
`cells, an observation that supports previous reports [15-17].
`preference to CD4* T cells (unpublished observations). Thus, the
`Of
`the £1 integrins, CD49d is expressed on resting
`lymphocytes whereas most of the other members of this family
`current findings demonstrate that the level of CD11a expression
`and activation is higher on CD8*Tcells than CD4* T