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Department of Pathology, University of Cambridge, Cambridge, United Kingdom; and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of nondepleting anti-CD4 Abs to induce tolerance in a primed immune system has been shown in several models of transplantation and autoimmunity (11, 14, 15, 16, 17, 18, 19). Much work has been conducted to establish the mechanisms by which such tolerance is achieved (reviewed in Ref. 20). It has been clearly demonstrated that control is maintained by a population of CD4+ T cells (21), and it has been suggested that this form of dominant or infectious tolerance may develop in the presence of the Ab by selectively promoting Ag-specific CD4 regulatory T cells at the expense of Th1 CD4 cells (reviewed in Ref. 22).
While the generation of regulatory or anergic T cells at the beginning of a response may be sufficient to control the development of pathological responses, the ability of nondepleting anti-CD4 to inhibit primed T cells so effectively requires some explanation. To clarify this, we have conducted a detailed examination of the effect of nondepleting anti-CD4 Abs on the transfer of diabetes by the CD4+ T cell clone BDC2.5 (23). This Th1 clone rapidly transfers diabetes to young NOD or NOD-scid mice, thus providing a model system to examine the role of anti-CD4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD and NOD/Lt-scid/scid mice were bred and maintained in the animal facilities of the Department of Pathology, University of Cambridge (Cambridge, U.K.). They received standard laboratory food and water ad libitum. NOD-scid mice were housed in microisolator cages and handled under sterile conditions in a laminar flow hood.
Antibodies
Monoclonal Abs YTS 177 (rat IgG2a anti-mouse CD4) (24) and YTH 34.5(rat IgG2a isotype control) (from Prof. H. Waldmann, Oxford University, Oxford, U.K.) were produced in vitro by growth of the hybridomas in a hollow fiber system. Abs were purified by precipitation with saturated ammonium sulfate and dialyzed extensively against PBS. The protein concentration was estimated by measuring the OD280. KT3 (CD3) and KT6 (CD4) (from Dr. K. Tomonari, Fukui Medical School, Fukui, Japan) and YTS 105 (CD8) (from Prof. H. Waldmann) were grown under standard tissue culture conditions, and spent supernatants were harvested for use in immunohistochemistry.
Propagation of BDC2.5
The diabetogenic T cell clone BDC2.5 was derived from the spleen and lymph nodes of newly diabetic female mice as described previously (23). Cultures were restimulated every 2 wk by combining 1 x 106 T cells, 2.5 x 107 APCs (irradiated NOD spleen cells), 10 µg ß cell membrane Ag (25), and human recombinant IL-2 (1000 U) in 20 ml of culture medium (IMDM) supplemented with 10% FBS. To expand cultures for the disease transfer experiment, the 20-ml volume containing the 4-day culture was transferred to a larger flask with 50 ml of new culture medium with more IL-2 (3500 U) for a further 4 days. T cells were washed and resuspended in sterile PBS for injection.
Disease transfer
For each experiment, litters of young NOD or NOD-scid mice (4–11 days old) were injected i.p. with 1x107 BDC2.5 T cells on day 0. Recipient mice were tested daily for glucose in the urine from day 4 onwards using Diastix (Bayer Diagnostics, Basingstoke, Hants, U.K.).
Administration of Abs
Timing of Ab administration may vary, and details are given in Results. In most experiments, neonates were treated i.p. with 1 mg of Ab three times a week for 3 wk, beginning either the day before or the same day as the transfer of BDC2.5.
T cell proliferation assay
Proliferation responses of the BDC2.5 cells to Ag in the absence of exogenous IL-2 were assayed by placing 5 x 104 T cells/well in triplicate in 96-well flat-bottom culture plates with 5 x 105 irradiated NOD spleen cells as APC and 10 µg ß cell membrane Ag. YTS 177 or control Ab YTH 34.5 was present at 100 µg/ml throughout the culture period. After 3 days, the cultures were pulsed with [3H]thymidine (1 µCi/well) and were harvested for counting after an overnight incubation.
IFN-
assays
For IFN- measurements, 5 x 104
BDC2.5 cells/well were placed in 96-well flat-bottom culture plates
with 5 x 105 irradiated NOD spleen cells as
APC and 10 µg ß membrane Ag. YTS 177 or control Ab YTH 34.5 was
present at 100 µg/ml throughout the culture period. Supernatants were
taken at 48 h and frozen at -20°C until assayed. As a control,
a CD8 CTL clone specific for influenza nucleoprotein (NP) (L. E. S.
Bowie, J. P. Tite, and A. Cooke, unpublished
observations) was assayed in a similar way using NP peptide as Ag.
IFN- was detected by sandwich ELISA as described earlier
(26) but using XMG.1 (rat anti-IFN-, grown and
purified in our own laboratory) at 6 µg/ml to coat the plates and
polyclonal rabbit anti-IFN- (Cambridge Biosciences, Cambridge,
U.K.) to detect.
Immunohistochemistry
Pancreases were removed at sacrifice and snap frozen in isopentane. Five-micrometer cryostat sections were air dried and fixed in acetone for 10 min. Air-dried sections were stored at -80°C.
Pancreatic ß cells were detected by preblocking sections with 20% normal mouse serum followed by incubation with guinea pig anti-porcine insulin (Dako, High Wycombe, U.K.) in 10% normal mouse serum and detected by rhodaminated goat anti-guinea pig IgG (ICN Pharmaceuticals, Thame, U.K.) in 10% normal mouse serum. NOD MHC class II was detected using OX-6 directly conjugated with FITC (Serotec, Kidlington, U.K.). Macrophages were detected in some experiments with monoclonal F4/80 directly conjugated with FITC (Serotec) and in others with polyclonal rabbit anti-F4/80 (provided by Prof. S. Gordon, Oxford University).
The T cell markers CD3, CD4, and CD8 were detected using appropriate mAb supernatants and visualized with FITC rabbit anti-rat Ig (Serotec).
CFSE labeling of cells
BDC2.5 cells were harvested 8 days after restimulation, centrifuged, and resuspended at 5x107 cells/ml in PBS. 5,6-Carboxyfluorescein diacetate succinyl ester (CFSE; Molecular Probes, Eugene, OR) was prepared as a 5-mM stock solution in DMSO. One microliter of the stock solution was added per milliliter of cells in PBS, and this was incubated at 37°C for 15 min. The incubation was stopped by addition of 10 ml of cold PBS, and the cells washed and resuspended in PBS for injection.
RNA extraction, cDNA preparation, and PCR
RNA from pancreases was obtained by guanidine thiocyanate extraction and ultracentrifugation over cesium chloride (27). One microgram of RNA from each sample was reverse transcribed to cDNA, and a standard amount of this was subjected to PCR (28). PCR conditions were 25–35 cycles of 30 s at 94°C, 1 min at 60°C, and 30 s at 72°C after an initial 5-min period at 94°C. Water controls and positive and negative controls for the relevant product were always run in parallel to the pancreas cDNA samples for the maximum number of cycles. The product was collected and stored at -20°C until required.
Three microliters of the highest cycle number product were subjected to electrophoresis on 2% agarose gels to check for the presence or absence of product of the correct size.
Primers for IL2, IFN-, inducible NO synthase (iNOS), Vß4, and
hypoxanthine phosphoribosyl transferase (HPRT) were used. The sequences
of the sense and antisense primers were as follows: IL2, sense 5'-TGA
TGG ACC TAC AGG AGC TCC TGA G-3'; antisense, 5'-GAG TCA AAT CCA GAA CAT
GCC GCA G-3'; IFN-, sense, 5'-TGG AGG AAC TGG CAA AAG GAT GGT-3';
antisense, 5'-TTG GGA CAA TCT CTT CCC CAC-3'; iNOS, sense, 5'-CCA CCT
TGT TCA GCT ACG CC-3'; antisense, 5'-GGA CAT CAA AGG TCT CAC AG-3';
HPRT, sense, 5'-GTA ATG ATC AGT CAA CGG GGG AC-3'; antisense, 5'-CCA
GCA AGC TTG CAA CCT TAA CCA-3'; Vß4 (primers for Vß4 were a
downstream primer complementary to the Cß region and an upstream
primer corresponding to the Vß4 region) 5'-GCA GGT CCA GTC GAC CCG
AAA AT- 3'; Cß, 5'-GGG TGG AGT CAC ATT TCT CAG ATC-3'.
Dot blot analysis of PCR product
Five-microliter volumes of PCR products were subjected to dot blot analysis by standard methods (18). Preblocked nylon membranes were reacted with 5 µl digoxigenin-labeled probe (labeling performed using a Boehringer kit and following the kit instructions; Boehringer Mannheim, Mannheim, Germany). After stringency washes, probe binding was visualized by incubation with an alkaline phosphatase-conjugated anti-digoxigenin Ab followed by a chemiluminescent substrate (CSPD; Boehringer Mannheim) and exposed to autoradiographic film (Hyperfilm ECL; Amersham Life Science, Little Chalfont, Bucks, U.K.).
Probes for the products are as follows: IL2, 5'-CAC CTT CAA ATT TTA CTT
GCC CAA GCA GGC C-3'; IFN-, 5'-GTG GAC CAC TCG GAT GAG CTC ATT-3';
HPRT, 5'-GCT TTC CCT GGT TAA GCA GTA CAG CCC C-3'; iNOS, 5'- CCA AAC
ACA GCA TAC CTG AAG GTG TG-3'; Vß4, the Vß4 primer was used as a
probe.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have shown that the monoclonal nondepleting
rat IgG2a anti-CD4, YTS 177, can prevent spleen cells from diabetic
NOD mice from transferring IDDM to irradiated NOD recipients
(11). We and others have been able to demonstrate that one
of the ways in which this Ab may generate and maintain peripheral
tolerance is through the generation of regulatory T cells
(15 , 21 ; and reviewed in Ref.
22). However, the ability of the Ab to inhibit highly
primed autoreactive cells suggested that the anti-CD4 may be able
to act directly on the effector T cell and not simply through the
indirect action of another T cell. To establish a simple system to
analyze this more fully, we initially examined the effect of YTS 177 on
the ability of the CD4+ T cell clone, BDC2.5, to
transfer IDDM to neonatal NOD mice. Fig. 1
shows that YTS 177 completely protects
the NOD neonates from diabetes induction by BDC2.5, whereas 90% of
control mice become diabetic. We have used a variety of control Abs,
including the YTH 34.5 used in the in vitro experiments below, on many
occasions and have never observed any effect of control Abs on the
ability of BDC2.5 to transfer IDDM. In more recent experiments, we have
found that 100 µg/dose rather than 1 mg of YTS 177 is sufficient, and
in another experiment a single dose of 300 µg completely protected
from diabetes (data not shown). Histological examination of the
pancreases from these mice (Fig. 1, H–J) shows that the mAb
treatment had not wholly prevented mononuclear cell infiltration into
the pancreas. In the pancreases of Ab-treated mice, there has clearly
been recruitment of CD8+ cells (data not shown)
and macrophages, and most notable is the up-regulation of MHC class II
expression, as detected by OX-6 Ab. Staining for CD4 is very faint,
suggesting that most of the CD4 has been down-modulated (data not
shown).
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As our prime objective was to determine whether there was a direct
action of YTS 177 on the Th1 clone, we transferred the BDC2.5 cells
into NOD-scid neonates. In this situation, the only T cells
available to the Ab were the committed effector cells. Fig. 2A shows that YTS 177 also
completely protected the NOD-scid neonates from diabetes by
BDC2.5 transfer. This confirms that the nondepleting Ab must be acting
directly on the primed cell population. Histological examination of the
pancreases from the Ab-treated mice showed that 3 days after the
transfer, T cell infiltration had occurred with recruitment of
macrophages and some up-regulation of class II MHC (Fig. 2, B–D). However, the degree of infiltration in the
NOD-scid neonatal recipients was less than in their
non-Ab-treated control counterparts and by 34 days had resolved to a
greater degree than in similarly treated NOD neonates (data not
shown).
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As fewer cells were present in the pancreas of YTS 177-treated
NOD-scid mice (data not shown), it was possible that the Ab
might be influencing the T cell trafficking. To examine this
possibility, YTS 177 administration was delayed until the T cells were
already present in the islet areas. Histological examination of the
pancreas showed that 1 day after the T cell transfer BDC2.5 cells had
already reached the islets, and 3 days after the transfer there was
significant islet infiltration with concomitant ß cell destruction
(Fig. 3, A–E). When the Ab
was administered at this time (i.e., from day 3), only a small number
(17%) of the recipients became overtly diabetic, as measured by
urinary glucose (Fig. 3F). The four control mice, depicted
in Fig. 3F, which were diabetic on day 6, were given YTS
177, and, interestingly, although these mice continued to register
positive for glucose in the urine, their condition did not deteriorate
further but they thrived. This was in marked contrast to our previous
studies, which have consistently shown that after transfer of BDC2.5
neonates that became diabetic quickly (by day 6) usually died within a
few days. Twenty-one days after the transfer of the clone, the mice
were killed, and pancreatic sections were examined histologically.
Almost all the T cell and macrophage infiltrate had disappeared as had
the MHC class II expression. We wondered if there had been some islet
regeneration either through neogenesis or replication of residual ß
cells. To assess whether there was any evidence of regeneration, the Ab
treated mice were given 5-bromo-2'-deoxyuridine 18 h before they
were killed. This allowed us to identify any dividing cells in the
pancreatic ducts and in the remaining islets (29). We
could not find any evidence for the regeneration of ß cells in these
mice (data not shown).
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, and iNOS in pancreases
of NOD-scid mice infiltrated with BDC2.5
Because administration of YTS 177 to diabetic mice appeared to
have an immediate impact on disease progression, we looked for an
effect of the Ab on cytokine mRNA expression within 24 h of Ab
treatment. A cohort of NOD-scid neonates was injected i.p.
with BDC2.5, and all tested negative for glucose in the urine on day 4.
On day 5, half of the neonates were given a single i.p. injection of 1
mg of YTS 177, and all were killed 24 h later. RNA was extracted
from the pancreas, reverse transcribed to cDNA, and subjected to PCR.
Fig. 4 shows the results of dot blot
analysis performed on the RT-PCR products. It is clear from the figure
that the densities of the dots for both HPRT and Vß4 are similar for
all samples. This indicates that the loading was similar for all
samples (HPRT) and that the numbers of BDC2.5 cells in the pancreatic
samples were similar (BDC2.5 TCR uses Vß4), thus confirming the
histology. However, mice that had been treated with YTS 177 showed
reduced expression of mRNA-encoding IL2, IFN-, and iNOS within
24 h of Ab treatment.
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production in an in
vitro proliferation assay with BDC2.5
To see if the reduced expression of mRNA for IFN- resulted in
reduced production of IFN-, we measured the levels in an in vitro
assay. Fig. 5 shows that YTS 177
completely inhibits production of IFN- by cultured BDC2.5 T cells in
response to Ag (Fig. 5B) and that proliferation of these
cells is also inhibited (Fig. 5A). As a control, we assayed
cells of a CD8 CTL clone specific for NP peptide (Fig. 5C),
and YTS 177 had no effect on the production of IFN- by this T cell
clone. This demonstrates that the Ab was working specifically on
CD4+ T cells.
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One feature of infiltration by the BDC2.5 clone is the
up-regulation of MHC class II on ductal epithelium. It is likely that
this is the result of inflammatory cytokines released by the T cells.
Because YTS 177 inhibits production of IFN- in vitro and reduces
message for IFN- in vivo, we looked for MHC class II expression on
the ducts after diabetic mice were treated with the Ab. Fig. 6A shows that when a mouse
first becomes diabetic there is significant up-regulation of MHC class
II expression (OX-6 stained) on all ducts as well as on other
surrounding tissue and infiltrating cells. However, 1 day after
administration of YTS 177 to a diabetic mouse, a small reduction in the
intensity of staining on the ducts could be seen (Fig. 6B),
and, 4 days after the Ab administration, most of the class II
expression on the ducts had gone (Fig. 6, C and
D). As stated previously, we find that if recipients of
BDC2.5 become diabetic within 6 days of the T cell clone transfer, they
do not survive for >2–3 days. This meant that we were unable to
examine a comparable control group. However, mice that became diabetic
17 days after transfer survived longer and were examined as many as 8
days later: there was no resolution of MHC class II expression (Fig. 6, E and F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies clearly show that YTS 177 is able to prevent the diabetogenic T cell clone from transferring IDDM and that disease inhibition can be achieved at a relatively late stage in the disease process. This inhibition is observed when neonatal NOD-scid, as well as neonatal NOD, recipients of the diabetogenic clone are treated with the Ab, thus demonstrating that it can be achieved in the absence of other T cells. By CFSE labeling the T cell clone, it has been possible to track its fate in vivo. The T cell clone can be found in the islet area within 24 h of cell transfer, and rapid recruitment of other cells such as T and B cells (in NOD recipients) and macrophages (in NOD and NOD-scid recipients) occurs. This coincides with marked up-regulation of expression of MHC class II on vascular endothelium and ductal epithelial cells adjacent to areas of inflammation. When anti-CD4 is injected, it is noticeable that this increased MHC expression diminishes rapidly, and, in NOD-scid recipients in particular, there is also a marked reduction in the extent of the pancreatic infiltration. The difference between NOD and NOD-scid recipients in the pancreatic infiltration may reflect the recruitment of other lymphocytes in the NOD neonates, which themselves contribute to the inflammatory process in the pancreas, increasing both its degree and longevity.
As MHC expression is up-regulated by cytokines such as IFN- and
TNF-
, one explanation for the effect of anti-CD4 on MHC
expression would be the early, dramatic effect that it clearly has on
IFN- mRNA levels. The decrease in both IFN- and iNOS mRNA levels
in the pancreas of YTS 177-treated mice suggests a reduction in
macrophage activation. It is probable that a CD4+
T cell clone such as BDC2.5 mediates ß cell destruction in
NOD-scid recipients through both its own cytokines and those
induced together with reactive oxygen intermediates in recruited
macrophages. As cytokines such as IL-1 inhibit insulin biosynthesis
(30), the inhibition of proinflammatory cytokine
production by YTS 177 and the concomitant cessation
of an inflammatory cascade may therefore represent one way by which the
autoimmune destruction of the ß cell is arrested. A corollary of the
inhibition of macrophage activation would be a reduction in IL-1 levels
and a restoration of insulin biosynthesis. In this context, it is
interesting to note that we observed increased levels of immunoreactive
insulin in the remaining ß cells when Ab treatment was started 3 days
after the transfer of BDC2.5. This suggests that those remaining ß
cells may be compensating for diminished circulating insulin
levels.
We explored the possibility that ß cell regeneration either through
cell division of remaining ß cells or neogenesis was occurring in the
tolerized mice. Our previous studies in spontaneously diabetic animals
had shown evidence of regenerative processes (29);
however, when we examined the pancreases of the YTS 177-treated mice we
found no evidence of ß cell regeneration. It is possible that the
inflammatory cytokines themselves were contributing to the increased
ductal cell proliferation and cell neogenesis that we had observed
in the ducts of diabetic mice. IFN- has been shown, in transgenic
mice, to be capable of eliciting such a regenerative process
(31).
We were able to follow the fate of BDC2.5 cells in vivo by using
CFSE-labeled cells and by staining sections with anti-CD3 and were
able to determine whether BDC2.5 cells remained in the pancreas
following Ab-mediated inhibition of ß cell destruction. The
transferred T cells could not be found in the pancreas of
NOD-scid mice 2 wk after Ab treatment nor were significant
numbers detectable in the spleens of treated mice. This suggests that
these T cells had been eliminated. The presence of apoptotic cells in
the pancreas was analyzed by TUNEL staining, but no clear difference
could be seen between anti-CD4-treated or control recipients of
BDC2.5 as significant levels of apoptosis were seen in both groups
involving both ß cells and lymphocytes. Although very few transferred
BDC2.5 cells could ever be detected in the spleen, it was possible to
recover Ag-reactive cells from NOD-scid spleens following
repeated restimulation with Ag in vitro showing that not all
transferred T cells had been eliminated. It is possible that the
remaining cells never gained access to islet Ag in vivo and they were
spared the effect of anti-CD4 treatment. It is interesting to note
that in vivo these cells could never mediate ß cell destruction as
YTS 177-treated NOD-scid recipients of BDC2.5 remain
normoglycemic indefinitely. This is consistent with the observation of
Scully et al. (32), who showed that for 4 wk after a
course of therapeutic Ab to induce tolerance to skin grafts, T cells
could be recovered from the spleens that were still competent to reject
grafts. It is likely that these T cells are protected from elimination,
as they are not at the site of the graft and therefore not undergoing
activation at the time of Ab treatment. Our in vivo data imply that T
cells undergoing activation are eliminated by the action of the
nondepleting anti-CD4. This observation was made in vitro by Newell
et al. (33), who showed that T cells underwent
activation-dependent cell death by apoptosis when a nondepleting
anti-CD4 Ab was added before stimulating through the TCRß.
In this paper, we have shown in vivo that YTS 177 has a direct effect on activated diabetogenic Th1 cells resulting in an inhibition of proinflammatory cytokines followed by elimination of the activated cells. Nondepleting Abs have no long-term effect on lymphocytes not activated at the time of their administration, so the demonstration that nondepleting anti-CD4 Ab can eliminate the activated effector CD4 cells makes this approach particularly attractive for autoimmune therapy where primed T cells are already active.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jenny Phillips, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
3 Abbreviations used in this paper: NOD, nonobese diabetic; CFSE, 5,6-carboxyfluorescein diacetate succinyl ester; HPRT, hypoxanthine phosphoribosyl transferase; IDDM, insulin-dependent diabetes mellitus; iNOs, inducible NO synthase; NP, nucleoprotein.
Received for publication January 27, 2000. Accepted for publication June 2, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transgenic mice. Development 118:33.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, n = 10), and the other group received
BDC2.5 plus Ab (
, n = 12). Disease was assessed
by measuring glucose in the urine. Mice were killed after 1 or 2
days of positive urine tests, and diabetic status was confirmed by
measuring blood glucose. Remaining mice, negative for glucose in the
urine, were killed 32 days after the transfer. B–G,
Snap-frozen pancreas sections from a representative neonate killed 10
days after transfer of BDC2.5, before the onset of overt diabetes.
B, C, and D are sequential
sections showing remnants of two islets (red fluorescence, insulin) and
infiltration of T cells CD3 (B), CD4 (C),
and CD8 (D). Other sections show macrophages
(F4/80+) (E), B cells (B 220+)
(F), and there was also up-regulation of MHC class II
(OX-6+) (G), particularly on ductal
epithelium. H–J, Mice in the YTS 177-treated group,
which were free of diabetes 32 days after the transfer, showed that
some infiltration had occurred with macrophages and up-regulated MHC
class II still present (red fluorescence, insulin; green fluorescence,
markers; magnification, x400).
, n = 6). Disease onset was assessed by
measuring glucose in the urine, and the four mice in the control group
that had developed IDDM were then also given the course of Ab. Mice
were killed 21 days after the transfer. Snap-frozen and stained
pancreas sections from mice treated with Ab on day 3
(I–K) and from mice treated on day 6 after becoming
diabetic (L–N) are shown (I–N, red
fluorescence, insulin; green fluorescence, markers; I
and L were stained for CD3 but were negative;
magnification, x400).
