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Division of Dermatology, University of Leicester, Leicester, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 chain of type I collagen, the
structure of which was confirmed by amino acid sequencing. The results
demonstrate the potential importance of type I collagen, an abundant
extracellular matrix protein, in enhancing the activation of
extravascular effector T cells in inflammatory disease, and point to a
new immunotherapeutic target. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits (CD49)
that influence ECMP-binding specificity (1, 2). A study of
T cell adhesion to ECMP demonstrated enhanced binding of freshly
purified human peripheral blood CD4+ T cells to
immobilized fibronectin and laminin, but not to type I collagen,
following a 10-min exposure to different activating stimuli
(2). These findings are supported by three reports
indicating that type I collagen, coimmobilized with anti-CD3,
causes only minor or no proliferation of freshly isolated, resting
peripheral blood T cells when compared with the effects of fibronectin
(3, 4, 5). In contrast, a further report suggested that
immobilized type I collagen could cause concentration-related
costimulation of freshly harvested peripheral blood T cells
(11). Others suggested that the collagen-induced T cell
responses might have been due to an indirect effect of contaminating
fibronectin (1), as fibronectin binding sites on collagen
are well described (12). Alternative explanations included
an altered state of T cell activation through purification by SRBC
rosetting (4), or possibly the methods used to immobilize
ECMP (5). Whatever the explanation for the contradictory
collagen-induced effects, attention in recent years seems to have been
focused mainly on the costimulation of resting T cells by fibronectin,
which was shown to be mediated specifically via VLA-4 and VLA-5
integrin heterodimers, incorporating 4 and 5 chains, respectively
(1, 2, 4, 5). The evidence that VLA-4 is a ligand for the
alternatively spliced connecting segment 1 domain of fibronectin, and
for VCAM-1, which both deliver costimulatory signals in T cells, has
identified this integrin as a therapeutic target. This has led to
multiple reports of the development and testing of potentially
therapeutic VLA-4 Abs and peptide ligands, both in vitro and in animal
models of inflammatory and autoimmune diseases, and transplantation
(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Early human studies with VLA-4 targeting agents
are also in progress, for example in patients with multiple sclerosis
(26, 27). The studies that demonstrated the costimulatory effects of immobilized fibronectin, but not type I collagen on T cells used resting PBL purified either by rigorous negative selection with immunomagnetic beads (4), or by a combination of plastic and nylon column adherence to remove monocytes and B cells, SRBC rosetting, and negative selection panning (5). We have considered that such freshly prepared, resting peripheral blood T cells are not representative of the populations of extravascular effector cells that will encounter immobilized ECMP and that are involved in the pathogenesis of inflammatory and autoimmune diseases. For example, in the skin lesions of atopic dermatitis, essentially all dermal T cells were found strongly to express the activation marker, HLA-DR, whereas less than 4% of peripheral blood T cells were HLA-DR+ (28). Similarly, in the skin lesions of psoriasis, more than 80% of dermal CD4+ T cells were HLA-DR+ (29). We have therefore conducted a detailed analysis of the costimulatory effects of ECMP on both freshly isolated, highly purified human peripheral blood CD4+ and CD8+ T cells, and highly purified Ag- or mitogen-stimulated CD4+ and CD8+ T cell lines used as models of the extravascular effector cells encountered in disease. We have confirmed that purified, resting peripheral blood T cells are costimulated by immobilized fibronectin and laminin, but not type I collagen. In contrast, we have shown that immobilized human type I collagen is a highly potent, ß1 integrin-dependent costimulator of the effector T cell populations, with a potency greater than that of fibronectin in the previously reported bioassays (4, 5) incorporating coimmobilized anti-CD3 mAb.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fibronectin from human plasma, laminin from human placenta,
collagen types I and III from human placenta, PHA, and Con A were
purchased from Sigma (Poole, U.K.). Stock solutions of the collagens
were made by redissolving the lyophilized preparations in 0.5 M acetic
acid at 1 mg/ml, and were stored at 4°C. Fibronectin was supplied as
a lyophilizate of 0.05 M TBS. Stock solution was prepared by diluting
this to 1 mg/ml in water, and stored in aliquots at -20°C. Laminin
was supplied in Tris-buffered NaCl at 0.5 mg/ml and was stored at
-80°C in aliquots. These storage conditions were as recommended by
the supplier. CD3 mAb OKT3 was from Cilag Biotech (High Wycombe, U.K.),
and CD29 mAb 4B4 from Beckman Coulter (High Wycombe, U.K.). Purified
mAbs against 1–5 integrins (clones FB12,
P1E6, ASC-1, P1H4, and P1D6, respectively), used mainly for flow
cytometry, were from Chemicon (Harrow, U.K.); FITC-conjugated mouse
anti-human CD4 and CD8 mAb and negative control Ab from Dako (Ely,
U.K.); and PE-conjugated goat anti-mouse IgG from Serotec (Oxford,
U.K.). Purified IgG1-blocking mAb AJH10 and 26F8, against
1 and 2 integrins,
respectively, were obtained from Dr. Roy Lobb (Biogen, Cambridge, MA).
Blocking mAb against 3 (P1B5; IgG1) and
4 (P4G9; IgG3) integrins, both in dialyzed
culture supernatant, were from Dako. Blocking mAb against
5 integrin (purified SAM-1; IgG2b) was from
Serotec. Purified, isotype control IgG3 and IgG2b mAb were from Dako,
and IgG1 was a gift from Dr. R. James, University of Leicester
(Leicester, U.K.). Tetanus toxoid was from Evans (Horsham, U.K.), and
rIL-2 from Eurocetus (Harefield, U.K.).
Preparation of fresh, resting human peripheral blood CD4+ and CD8+ T cells
PBMC were obtained from citrate anticoagulated blood of healthy adult volunteers by standard density-gradient centrifugation over Lymphoprep (Life Technologies, Paisley, U.K.). Interface PBMC were pelleted, washed, and resuspended in Earle’s balanced salt solution at 4°C. For purification of CD4+ T cells, PBMC were resuspended in 1 ml Earle’s balanced salt solution with Dynabeads M-450 CD4 (Dynal, Bromborough, U.K.) at a ratio of beads to target cells of 4:1. Dynabead-bound T cells were separated and thoroughly washed in accordance with the manufacturer’s instructions, then resuspended in 100 µl RPMI 1640 medium containing 10% pooled human AB serum, 3 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (RH10) with an excess (1 U) of Detachabead M-450. Dynabeads were subsequently removed, and the remaining cells depleted of any contaminating CD8+ T cells by negative selection with M-450 CD8 Dynabeads. The resulting CD4+ population was >98% pure and contained <1% CD14+ monocytes, as determined by flow cytometry. Similarly, CD8+ T cells were positively selected from density gradient-purified PBMC by using Dynabeads M-450 CD8 and subsequent treatment with Detachabead M-450. Any contaminating CD4+ T cells were removed using Dynabeads M-450 CD4, the resulting CD8+ population also showing >98% purity by flow cytometry.
Preparation of CD4+ and CD8+ T cell lines
Peripheral blood CD4+ cells, highly purified with Dynabeads as described above, were used to generate T cell lines. Autologous irradiated PBMC and the purified T cells (2:1 ratio) were cultured in RH10 with IL-2 (100 U/ml) and tetanus toxoid (5 µg/ml). At least three rounds of stimulation with Ag and irradiated PBMC were done at 10-day intervals. Cultures were supplemented with fresh medium and pulsed with IL-2 (100 U/ml) every 3–4 days. Following the last round of Ag stimulation, cells were pulsed with 20 U/ml IL-2 after 7 days, then maintained in culture for an additional 3 days before storage under liquid nitrogen. Before use in bioassays, the cells were thawed, washed in RH10, and then used immediately. Dynabead-purified CD8+ peripheral blood T cells were used to generate CD8+ cell lines exactly as for the above CD4+ protocol, except that tetanus toxoid was replaced with 2 µg/ml PHA (30). The respective lines were shown by flow cytometry to consist of >98% CD4+ or CD8+ cells.
Flow cytometric analysis
T cells (5 x 105 in 100 µl PBS
containing 2% BSA) were incubated with ß1 or
integrin mAb, IgG1, or IgG3 isotype controls (all 2 µg/ml),
followed by PE-conjugated goat anti-mouse IgG (1/10 final
dilution). Cells were also labeled with FITC-conjugated mouse
anti-human CD4 or CD8 mAb (10 µg/ml) before analysis by FACScan
(Becton Dickinson, Oxford, U.K.).
T cell proliferation assays
Anti-CD3 mAb OKT3 (0.5 µg/ml in PBS for experiments with
fresh, resting PBL as in Fig. 1
, A and B, and 0.1
µg/ml for all other experiments) was placed in microtiter plate wells
and incubated overnight at 4°C. After three washes with PBS to remove
unbound mAb, different amounts of ECMP were added for 2–3 h at room
temperature (4). Unbound ECMP was removed by three washes
with PBS. T cells (5 x 104/well) were then
added in RH10 or AIM V serum-free medium (Life Technologies), and
incubated for 3 days. During the last 8 h of the assay, cultures
were pulsed with 1 µCi/well [3H]TdR, T cells
were harvested, and incorporated radioactivity was counted. In certain
proliferation assays, freshly purified CD4+ T
cells from peripheral blood or tetanus toxoid-responsive
CD4+ T cell lines (5 x
104/ml) were incubated with twice the number of
irradiated, autologous PBMC, and appropriate concentrations of tetanus
toxoid in ECMP-coated or uncoated microtiter plate wells for 3–5 days,
and [3H]TdR uptake determined. In other
experiments, mAb that blocked integrin function (up to 10 µg/ml), and
isotype controls were added to 3-day proliferation assays with T cell
lines, coimmobilized anti-CD3 (0.1 µg/ml), and either fibronectin
(10 µg/ml) or type I collagen (1 µg/ml). In experiments to test the
effects of the ß1 integrin-specific mAb 4B4 in
ECMP-free assays, a CD8+ T cell line was cultured
in the presence of 50–200 U/ml IL-2 and up to 2 µg/ml 4B4 for 3 days
in microtiter wells coated with anti-CD3 (0.1 µg/ml), and
[3H]TdR uptake determined. Results from all T
cell proliferation assays are expressed as mean cpm from triplicate
cultures.
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Stock solutions of type I collagen were purified in 100-µg
aliquots by sequential reversed phase HPLC on a 4.6 x 100-mm
Brownlee Aquapore RP-300 C8 column and a 4.6 x 250-mm Atlantis 5
µ C18 300 Å column (Phenomenex, Macclesfield, U.K.), both eluted at
1 ml/min with 0.1% trifluoroacetic acid in water for 5 min, followed
by a linear gradient to 0.1% trifluoroacetic acid in acetonitrile over
30 min. UV-absorbing peaks (215 nm) were collected manually,
evaporated, and tested for T cell costimulatory activity. Aliquots of
purified peptides were analyzed by SDS-PAGE on 6% acrylamide minigels.
Following staining with Coomassie blue, a 
100-kDa band corresponding
to the 1 chain of type I collagen was excised and trypsinized by
methods established in the University of Leicester Protein and Nucleic
Acid Chemistry Laboratory. Briefly, the band was destained repeatedly
in ethanol/50 mM ammonium bicarbonate (2:3, v/v), dehydrated by washing
in acetonitrile followed by vacuum desiccation, rehydrated in 10 mM
DTT/100 mM ammonium bicarbonate, dehydrated once again, and digested by
incubation with trypsin (75 µg/ml in 25 mM ammonium bicarbonate) at
37°C overnight. Resulting peptides were extracted from the gel into
5% trifluoroacetic acid in acetonitrile/water (1:1, v/v), and
following evaporation the residue was purified on a 1 x 250-mm
C18 Aquapore RP-300 HPLC column eluted with an acetonitrile gradient.
Residue from a peak eluting at 25% acetonitrile was subjected to
N-terminal amino acid sequencing in an ABI 476 liquid-phase protein
sequencer (Applied Biosystems, Foster City, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly purified, unstimulated peripheral blood
CD4+ and CD8+ T cells did
not proliferate in the presence of immobilized anti-CD3 or ECMP
alone (data not shown), but underwent substantial proliferation when
cultured in microtiter wells coated with anti-CD3 and fibronectin,
as found previously (3, 4, 5). Proliferation was less
pronounced with laminin coating, and only minimal or absent
with collagen types I or III (Fig. 1
, A and B), thus confirming two previous reports
(4, 5). In contrast, CD4+ and
CD8+ T cell lines proliferated strongly in the
presence of coimmobilized anti-CD3 and collagen type I, with the
effects of fibronectin, laminin, and collagen type III apparently being
less potent that those of type I collagen on a µg/ml basis. Thus, a
low coating concentration of type I collagen (0.6 µg/ml) caused
marked CD4+ and CD8+ T cell
costimulation, whereas the same coating concentration of fibronectin
and laminin gave small responses. Collagen type III gave intermediate
responses at this concentration (Fig. 1, C and
D). The greater potency of collagen type I than fibronectin
in costimulating a CD8+ T cell line was
demonstrated in further assays incorporating immobilized anti-CD3
and a wider range of concentrations of fibronectin and collagen type I.
In these assays, the concentrations of collagen type I and fibronectin
causing half-maximal responses were 0.04 µg/ml and at least 2.5
µg/ml, respectively, indicating at least 60-fold greater potency for
collagen type I in this system, on a µg/ml basis (Fig. 1E). The higher concentrations of type I collagen were
associated with apparently decreasing responses, as also seen in Fig. 1, C and D. The mechanism(s) responsible for this
bell-shaped dose-response curve has not yet been investigated.
Activation of T cell lines by the collagen type I was also shown to be
independent of serum factors such as fibronectin, as T cell
proliferation was not reduced in costimulation assays with AIM V
serum-free medium vs RH10 (Fig. 2, A and B).
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Preliminary experiments determined the threshold concentrations of
tetanus toxoid that caused proliferation of either freshly purified
peripheral blood CD4+ T cells, or
CD4+ T cell lines, to levels just above
background in 5- or 3-day assays, respectively, with autologous,
irradiated PBMC as APC (data not shown). Coating of microtiter wells
with type I collagen or fibronectin was subsequently shown to cause
marked enhancement of proliferation of the CD4+
line cultured with irradiated PBMC and the threshold amount of tetanus
toxoid, coating concentrations of ECMP as low as 20 ng/ml, causing
maximal costimulation (Fig. 3B). In this system, the
µg/ml potencies of type I collagen and fibronectin were similar. In
contrast and in line with results obtained in initial experiments with
immobilized anti-CD3 (Fig. 1A), 5-day assays with
freshly purified peripheral blood CD4+ T cells,
irradiated autologous PBMC, and a threshold concentration of tetanus
toxoid showed enhancement of proliferation by fibronectin, but not type
I collagen (Fig. 3A).
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1, 2, and
3 integrins on T cell lines, and integrin dependence of
type I collagen-induced responses
Two-color flow cytometry was used to determine the percentage of T
cells expressing specific integrins (Table I). As shown, the percentages of resting,
freshly purified peripheral blood CD4+ cells
expressing 1, 2, and
3 integrins were generally low, but expression
of these integrins by the corresponding T cell line from the same donor
was greatly enhanced. A similar trend was seen with
CD8+ cells. The percentage of expression of
4 and ß1 integrins was
high for both the resting peripheral blood cells and the T cell lines.
The percentage of expression of 5 integrin by
the freshly purified, resting cells was lower than for
4, but was enhanced on the T cell lines. The
previously reported bimodal expression of ß1
integrin on freshly harvested, resting CD4+ T
cells (4, 31) was confirmed in the present experiments,
was less pronounced on the resting CD8+ cells,
but was seen on both CD4+ and
CD8+ T cell lines (data not shown).
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, A and B). That
this effect of 4B4 mAb was not due to nonspecific cytotoxicity or
negative signaling via ß1 integrin was shown by
the lack of any inhibitory effect of 4B4 on a
CD8+ T cell line proliferating in ECMP-free
conditions, in the presence of immobilized anti-CD3 and three
different concentrations of IL-2 (Fig. 4C).
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integrin-specific
blocking mAb. As previously reported (4, 5),
fibronectin-induced costimulation of T cells was reduced by mAb against
VLA-4 or VLA-5 integrin (mAb P4G9 or SAM-1, respectively, in the
present experiments), and completely blocked by a combination of the
two (Fig. 4D). In contrast, type I collagen-induced
costimulation of a CD8+ T cell line was
unaffected by VLA-4 or VLA-5 mAb or a combination of these, but was
partially blocked by VLA-1 or VLA-2 mAb (AJH10 or 26F8, respectively),
with more pronounced inhibition by a combination of the two mAb (Fig. 4E). The VLA-3 mAb ASC-1 (Chemicon) is reported by the
manufacturers not to inhibit cell adhesion to fibronectin. However, we
found that it inhibited both fibronectin- and type I collagen-induced T
cell costimulation (data not shown), suggesting uncertain
integrin-blocking specificity. Similarly, the VLA-3 mAb P1B5 (Dako)
inhibited both fibronectin- and type I collagen-induced T cell
costimulation, both before and after dialysis (the mAb was supplied in
15 mM sodium azide). P1B5, both untreated and dialyzed, also inhibited
T cell proliferation in ECMP-free assays containing IL-2 and
anti-CD3 (data not shown), again suggesting uncertain
integrin-blocking specificity. Evidence for VLA-3 involvement in the
collagen-induced responses has therefore not been established. Purification and structural analysis of type I collagen
The type I collagen preparation (Sigma) used in this work was
prepared by acidic pepsin digestion of human placental homogenate, and
differential salt precipitation (32). Reversed phase HPLC
of this preparation on a 4.6 x 100-mm C8 column, under the
conditions described in Materials and Methods, demonstrated
a product of substantial purity (peaks 1 and 2, Fig. 5A). Repurification of each
peak on a 4.6 x 250-mm C18 column yielded separated peaks (1a and
2a, Fig. 5A). Following Micro bicinchoninic acid protein
assay (Pierce and Warriner, Chester, U.K.), the ability of equivalent
amounts of protein from each peak to stimulate a T cell line in the
presence of coimmobilized anti-CD3 was determined. This showed that
peak 1a contained the main stimulatory material (Fig. 5C).
SDS-PAGE of the unpurified type I collagen preparation showed a major
Coomassie blue-stained band at about 100 kDa and a less intense band at
about 95 kDa, compatible with the 1 and 2 chains of type I
collagen, which are present at a ratio of 2:1 (33). Higher
molecular mass bands were also present, suggestive of dimeric and
trimeric forms (Fig. 5B, lane 1). SDS-PAGE of
peak 1a (Fig. 5A) showed a single, major Coomassie
blue-stained band at 100 kDa, indicative of the 1 chain of type I
collagen, and less prominent higher molecular mass bands (Fig. 5B, lane 2). SDS-PAGE of protein from peak 2a
(Fig. 5A) showed a major band at 95 kDa, indicative of
the 2 chain of type I collagen, with a second, fainter band at
100 kDa indicating carry-over from peak 1, and prominent higher
molecular mass bands (Fig. 5B, lane 3). Following
SDS-PAGE of a larger quantity of peak 1a, the 100-kDa band was
excised and subjected to trypsin digestion, and N-terminal sequences of
two HPLC-purified peptides were obtained. These showed the sequences
Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg and Gly Pro Ala Gly Pro
Gln Gly Pro Arg Gly, which are unique to the 1 chain of type I
collagen, and not found in any other known protein, including multiple
other collagens.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 chain of type I collagen following analysis of a trypsin
digest. A synergistic effect of fibronectin in the human serum used
during culture could also be excluded as the serum-free medium, AIM V,
supported type I collagen-induced costimulation. In addition, positive
selection of fresh, resting PBL with immunomagnetic beads did not lead
to an altered state of activation sufficient to induce responses to
collagen. The responsiveness of the effector T cell populations to type I collagen was ß1 integrin dependent, as shown by the highly potent inhibitory effects of 4B4 mAb. This effect of 4B4 mAb was not due to nonspecific toxicity or negative signaling, as the Ab had no effects on a T cell line proliferating under ECMP-free conditions. The type I collagen-induced responses of the T cell lines in the present experiments are largely due to VLA-1 and VLA-2 integrins, as shown by their up-regulation on T cell lines and the effects of specific blocking mAb. As the blocking specificities of available VLA-3 mAb were uncertain, no sound evidence for involvement of this integrin was found.
The CD4+ effector T cell costimulatory properties
of type I collagen were supported by the findings in a more
physiological in vitro system in which immobilized anti-CD3 was
replaced by a threshold concentration of tetanus toxoid Ag and
irradiated autologous PBMC as APC. In these assays, a coating
concentration of type I collagen as low as 20 ng/ml was associated with
near maximal costimulation of the CD4+ tetanus
toxoid-responsive T cell line. While these findings do not exclude an
activating effect of type I collagen on APC populations, and thus
additional, indirect enhancement of T cell activation, a sole effect on
APCs alone is unlikely, as the irradiated PBMC were incapable of
activating freshly isolated, resting T cells in the presence of
immobilized collagen and the threshold concentration of Ag (Fig. 3A). Interestingly, the µg/ml potency of fibronectin in
costimulating the T cell line was similar to that of type I collagen in
this system, unlike the findings in the model system incorporating
immobilized anti-CD3. However, the inability of type I collagen to
costimulate freshly harvested, resting CD4+ PBL
in the presence of irradiated autologous PBMC and the threshold
concentration of tetanus toxoid directly reflects the results obtained
in the assays with coimmobilized anti-CD3.
The findings highlight the potential importance in T cell activation of
collagenous tissues such as the skin, in which 80% of collagen is type
I (33). The results imply that therapeutic interventions
targeted at interactions between T cells and ECMP (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
will be only partially effective in collagenous tissues if they do not
block the costimulatory effects of type I collagen. This possibility is
supported by the reports that VLA-4-blocking peptide and mAb are unable
to inhibit cutaneous contact hypersensitivity, delayed-type
hypersensitivity, or adjuvant arthritis in rodents by more than
35–60% (14, 15, 20, 25). This implies cell recruitment
and costimulation via additional mechanisms, including possibly those
mediated by type I collagen. The findings suggest that broader spectrum
inhibitors targeted at the ß1 subunit will be of greater therapeutic
value than specific integrin antagonists, when more robust
immunosuppression is required. Therapy targeted specifically at
interactions between T cells and type I collagen will also be of
interest, although it is currently not known whether RGD or other
collagen recognition sequences (34) are involved.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard D. R. Camp, Division of Dermatology, University of Leicester, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, U.K.
3 Abbreviations used in this paper: ECMP, extracellular matrix protein; VLA, very late Ag..
Received for publication January 27, 2000. Accepted for publication August 4, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chains. J. Biol. Chem. 265:19230.-4 subunit inhibit the murine contact hypersensitivity response. Eur. J. Immunol. 23:682.[Medline]
4ß1 interaction with connecting segment 1 and vascular cell adhesion molecule. J. Biol. Chem. 269:18668.4 integrins in vivo. J. Clin. Invest. 94:1722.
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) space available to cyclic pentapeptides. Eur. J. Biochem. 242:352.[Medline]
4 integrin antagonist peptide. J. Immunol. 158:4180.[Abstract]
4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J. Clin. Invest. 100:3083.[Medline]
4 integrin antibody on brain lesion activity in MS. Neurology 53:466.1(V)2(V)3(V) collagen molecule. J. Biol. Chem. 259:14170.2ß1 integrin in collagen. J. Biol. Chem. 266:7363.This article has been cited by other articles:
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) and 10 µg/ml (
). Proliferation of
CD4+ and CD8+ T cell lines is shown in
C and D, respectively, in which the
coating concentrations of ECMP in the different groups of columns are,
from left to right, 0.6 µg/ml, 1.25
µg/ml, 2.5 µg/ml, 5 µg/ml (C and
D), and in addition 10 and 20 µg/ml
(C). Similar results to those in A–D
were obtained in at least one more set of independent triplicate
experiments. FN, Fibronectin; LN, laminin; COL I, type I collagen; COL
III, type III collagen. In E, the effects of a wider
range of coating concentrations of type I collagen (
) on the proliferation of a CD8+ T cell
line are shown. All points represent the mean results of triplicate
cultures.
