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Department of Immunology and Barbara Davis Center for Childhood Diabetes, 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 BDC-2.5 TCR transgenic (Tg) mouse (26) was made with
the TCR 
-chain (V1) and ß-chain (Vß4) of the diabetogenic T
cell clone BDC-2.5, which was produced in our laboratory
(12). This NOD-derived T cell clone proliferates and makes
Th1 cytokines in vitro in response to whole islet cells or membrane
fractions obtained from ß tumor cells (13, 27). In vivo,
it rapidly and reproducibly transfers diabetes into young (<2-wk-old)
NOD or NOD.scid recipients but not into adult mice. As
previously described (28) and confirmed by studies in this
lab (C. Dobbs and K. Haskins, manuscript in preparation), T cells from
the BDC-2.5 TCR Tg mouse bred onto the NOD.scid background
can rapidly transfer diabetes at very low cell numbers (<1 x
106) into adult NOD.scid
mice.
Evidence from several labs has indicated that there is a protective, regulatory role in autoimmune diabetes for the IL-4/IL-10-secreting subset of CD4+ T cells, which are generally referred to as Th2 T cells. Treatment of NOD mice with IL-4 gave rise to IL-4-secreting spleen cells and delayed diabetes onset (29) and, in subsequent studies, protection by IL-4-secreting splenic T cells was shown to be transferable (30). Inserting the IL-4 gene under the rat insulin promoter on the NOD background was also found to be protective (31). Similar protective results were seen after systemic treatment of NOD mice with IL-10 (32). On the other hand, it has been reported that IL-4-secreting T cells obtained from a 4-day culture of spleen cells from the BDC-2.5 TCR Tg mouse could not protect against disease transfer (33) and, in a subsequent study, short-term (7-day) cultures of CD4+ T cells with IL-4 were found to induce diabetes in NOD.scid recipients (34).
In an attempt to resolve the discrepancies in the literature with respect to protective vs diabetogenic properties of IL-4-secreting T cells and to determine whether protective Th2 T cells could in fact be obtained from a mouse bearing the TCR of a diabetogenic Th1 T cell clone, we have investigated the in vivo properties of long-term T cell lines and clones with stable Th2 cytokine-secreting profiles from the BDC-2.5 TCR Tg mouse. Our results indicate that IL-4/IL-10-secreting CD4+ T cell lines bearing the transgenic TCR, like the original clone BDC-2.5, can rapidly cause diabetes in young NOD recipients but cannot induce disease in NOD.scid mice of any age.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of NOD and NOD.scid mice were obtained from either The Jackson Laboratory (Bar Harbor, ME) or the breeding colony at the Barbara Davis Center. BDC-2.5 TCR Tg breeding pairs were obtained from Dr. Jonathan Katz (Washington University, St. Louis, MO). Mice were bred and housed under specific pathogen-free conditions in the University of Colorado Health Sciences Center’s Center for Laboratory Animal Care. NOD and NOD.scid mice were used before they reached 15 days of age as recipients in transgenic T cell clone transfers. NOD mice (8–10 wk old) were used as a source of APC and fresh islet cells.
Isolation and maintenance of Tg T cell lines and clones
To produce Th2 T cell lines from the BDC-2.5 TCR Tg mouse,
splenic T cells (1 x 107/ml) from an
untreated, nondiabetic female donor were incubated with islet cells or
ß cell membranes (prepared from islet cell tumors) in a primary
culture for 5 days before being combined in a 20-ml secondary culture
in high-glucose DMEM (Life Technologies, New Island, NY) (supplemented
with 44 mM sodium bicarbonate, 0.55 mM L-arginine, 0.27 mM
L-asparagine, 1.5 mM L-glutamine, 1 mM sodium
pyruvate, 50 mg/L gentamicin sulfate, 50 µM 2-ME, 10 mM HEPES, and
10% FCS) with islet Ag, APC (2.5 x 107
irradiated NOD spleen cells), 3.5 U/ml IL-2 (IL-2-containing EL4
supernatant), 5 ng/ml IL-4 (X63-IL-4 supernatant) (35),
and anti-IFN-
mAb (XMG1.2) at a concentration of 2.5% v/v.
Islet Ag for these cultures was in the form of either fresh islet cells
(5 x 103) or 12 µg of a membrane
preparation made from ß tumor cells (27). Restimulation
cultures were incubated upright in 25-cm2 flasks
at 37°C and 10% CO2 for 2 wk. Tg T cell lines
were maintained thereafter on 2-wk cycles with fresh Ag, syngeneic APC,
IL-2, IL-4, and anti-IFN- mAb. Although T cell cultures from the
BDC-2.5 TCR Tg mouse were, as expected, heavily skewed toward T cells
of the BDC-2.5 phenotype, if these lines were cloned early, distinct T
cell clones with different properties could be isolated. Tg T cell
lines were cloned by limiting dilution in 96-well round-bottom plates
containing 5 x 105 APC, 5 x
103 islet cells or 1 µg of ß cell membranes
as Ag, IL-2, IL-4, and anti-IFN- mAb in each well. A summary of
Th1 and Th2 T cell clones used in this study is shown in Table I
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Expansion cultures for in vivo transfers were produced by culture of
3–6 x 106 T cells from 4-day restimulation
cultures with 7 U/ml IL-2 and 5 ng/ml IL-4 in 60 ml of supplemented
DMEM in 75-cm2 flasks for 4 days at 37°C and
10% CO2. T cells were harvested, washed three
times, resuspended in HBSS, and injected into young recipients.
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At the end of each 2-wk growth cycle, Tg T cell lines and clones were assayed for Ag specificity and cytokine production. To assess Ag specificity, T cells were plated in 96-well flat-bottom plates at 2 x 104 cells/well alone, with 2.5 x 104 syngeneic peritoneal cells as APC to determine nonspecific proliferation, or with 2.5 x 104 syngeneic peritoneal cells as APC plus 5000 irradiated NOD islet cells, or with 10 µg of ß-membrane, to determine Ag-specific proliferation. Plates were incubated at 37°C and 10% CO2 for 3 days. Tritiated thymidine was added at a concentration of 0.5 µCi/well for the final 6 h of culture. Assays were harvested on a Packard (Meriden, CT) Filtermate 196 harvester and read on a Packard Topcounter.
Cytokine production was assessed by ELISA analysis of Con A-stimulated
supernatants. Briefly, Tg T cells were plated at 4 x
104 cells/well in 96-well flat-bottom plates with
5 x 105 irradiated syngeneic spleen cells
as APC and Con A (Sigma, St. Louis, MO) at a final concentration of 2.5
µg/ml. Plates were incubated at 37°C and 10%
CO2, and supernatants were harvested at 48
h. Cytokine production by Tg T cell lines and clones was tested at the
end of a 2-wk growth cycle and from expansion flasks on the day of
injection for adoptive transfer experiments. Cytokine levels were
determined by specific sandwich ELISAs for the following cytokines:
IL-2, IL-4, IL-6, IL-10, and IFN-. Cytokine ELISAs were performed
using purified mAb (PharMingen, San Diego, CA) for capture and
biotinylated mAb (PharMingen) for detection and then using
streptavidin-HRP (Sigma) and
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) as the
substrate. Assays were read on a Titertek (Huntsville, AL) Multiskan
Plus platereader at 405 nm.
Confirmation of Tg TCR phenoype or genotype
Tg T cell lines were assessed for the expression of the BDC-2.5
TCR chain Vß4 by flow cytometry using a Vß4-specific mAb, KT4-10.
The presence of the V1 and Vß4 transgenes was confirmed by PCR
analysis using transgene-specific primers.
In vivo transfer of Tg T cell lines and clones
Recipients of Tg T cell lines were young NOD or NOD.scid mice 3–14 days of age. Recipients were given two i.p. injections of 1 x 107 cells 1 wk apart. Recipients were monitored for diabetes by daily urine (Diastix, Bayer, Elkhart, IN) or blood glucose (Glucotrend, Boehringer Mannheim, Indianapolis, IN) tests starting at 6 days postinjection until they became diabetic or the experiment was terminated. Overt diabetes was defined as a positive urine glucose (>1%) and then a positive blood glucose test of >250 mg/dl (14 mM). Recipients were sacrificed when blood glucose readings were 320 mg/dl (18 mM) or higher. At sacrifice, the pancreata were removed for histological analysis.
Histology
At sacrifice, pancreata were removed and placed in formalin for at least 24 h. Pancreata were subsequently embedded in paraffin, sectioned, and stained with aldehyde fuchsin (A/F). Histological sections were read visually by two independent scorers to determine the extent of islet infiltration and granulation of the islets. Infiltration was scored as follows: 0, no infiltrate; 1, mild peri-islet or polar infiltrate; 2, moderate peri-islet or mild intraislet infiltrate; and 3, severe intraislet infiltrate. Granulation was also scored from 0 to 3 by the following criteria: 3, 75–100% granulated islets; 2, 50–75% granulated islets; 1, 20–50% granulated islets; and 0, complete degranulation.
Cotransfer of Tg Th2 (Tg/T2) line with diabetogenic Th1 clones or diabetic spleen cells
For cotransfer experiments using diabetic spleen cells,
recipient NOD.scid mice (9–14 days of age) were injected
i.p. on day 0 with 2 x 107 diabetic spleen
cells, 1 x 107 of the transgenic T cell
line 2.5 Tg/T2-X, or spleen cells and 2.5 Tg/T2-X together. To obtain
spleen cells, diabetic NOD female donors were sacrificed and their
spleens were removed under sterile conditions. Spleens were ground into
a single-cell suspension using a tissue homogenizer; the cells were
washed twice with HBSS and counted using a hemacytometer. Splenocytes
were resuspended in HBSS at a concentration of 4 x
108 cells/ml, and 2 x
107 cells were injected into mice in a volume of
50 µl. For cotransfers involving a CD4+ T cell
clone, we used the T cell clone BDC-6.3, another Th1 diabetogenic T
cell clone from our original panel (13). This T cell clone
is Vß4 V3 in its TCR and proliferates to the same islet Ag
preparations as BDC-2.5 but, unlike BDC-2.5, cannot transfer disease to
NOD.scid mice of any age. Cotransfer experiments were
performed by injecting NOD.scid recipients (7–14 days of
age) i.p. on days 0 and 7 with 1 x 107 of
BDC-6.3, 1 x 107 2.5 Tg/T2-X, or BDC-6.3
and 2.5 Tg/T2-X together. All T cell clones were prepared in expansion
cultures as described previously. Recipients were monitored as
described above. After diabetic animals were sacrificed, their
pancreata were placed in formalin and histologically examined as
described previously.
Statistical analysis
Statistical significance within experiments was determined using JMP analysis software (SAS Institute, Cary, NC). Survival analysis was done using the product-limit (Kaplan-Meier) method. The endpoint of the experiment was defined as diabetes. Data on animals that did not become diabetic by the end of the experiment were censored. The p values shown were determined by Log-Rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell lines were generated from spleen cells obtained from
nondiabetic female BDC-2.5 TCR transgenic mice and cultured in the
presence of NOD islet Ag and IL-2. Lines were pushed toward a Th2
phenotype by the addition of IL-4 and anti-IFN- mAb. Tg/T2 lines
were selected on the basis of specificity for islet cells and
production of IL-4, and the absence of production of IFN-.
Subsequently, the Tg/T2 lines were extensively analyzed for cytokine
production, with testing for IL-2, IL-4, IL-6, IL-10, and IFN-. As
these lines, coming from TCR transgenic animals, were comprised
primarily of BDC-2.5 Tg-positive T cells, uncloned lines tended to
become clonal with continued culture. However, some lines were cloned
by limiting dilution shortly after being established. Each line or
clone selected showed a reproducible response to islet cell Ag above
background in routine T cell proliferation assays, although the
magnitude of the proliferative response was somewhat less than that of
the parent Th1 clone, BDC-2.5 (data not shown). All of the Tg/T2 lines
and clones were shown to bear the BDC-2.5 TCR by flow cytometry or PCR
analysis (data not shown).
As shown in Table II, two TCR Tg lines
and one clone appeared to have a Th2 phenotype. Every Tg/T2 line or
clone showed a distinct but consistent cytokine profile, producing
different levels of the Th2 cytokines IL-4, IL-6, and IL-10. The T cell
clone 2.5 Tg/T2-1.D6 made no IL-10 detectable by ELISA and, like
BDC-2.5, produced small but variable amounts of IL-2 (data not shown),
but as described below, the in vivo activity of this clone was the same
as the lines 2.5 Tg/T2-2 and 2.5 Tg/T2-X.
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To characterize the in vivo properties of the Tg/T2 lines and
clones, clone transfer experiments were conducted in young NOD
recipients. Recipient mice less than 14 days old were injected i.p.
with Tg T cell lines or clones (1 x 107
cells/injection). The diabetogenic Th1 T cell clone BDC-2.5 was used as
a positive control. With the two lines and the clone 2.5 Tg/T2-1.D6,
these transfers resulted in a rapid onset of diabetes in recipient
mice, with hyperglycemia developing by 14 days posttransfer (Table III). Numbers of animals in each
experiment were small because transfers in each case were with two or
three test clones (plus controls) into unweaned litters of mice. The
kinetics of disease onset were not significantly different from those
caused by the parent clone BDC-2.5.
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B. In some cases, the damage
was so extensive that a loss of pancreatic structure resulted. This
pattern of destruction was very similar to the damage caused by the
parent clone BDC-2.5 (Fig. 1A). In addition, pancreatitis,
or swelling of the exocrine tissue, was often observed in mice
receiving diabetogenic Tg/T2 cells, a result not usually seen with
BDC-2.5.
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Next, we determined the ability of the Tg/T2 lines and clones to
transfer disease to the young NOD.scid mice. As indicated in
Table IV, young NOD.scid
recipients rapidly become diabetic upon transfer of our islet-specific
Th1 clone BDC-2.5 but developed no disease with the Tg/T2 lines or
clones with either one or two injections of 1 x
107 cells. Again, numbers of animals in
individual experiments were small due to transfers having to be
performed in unweaned litters of mice. Histological analysis revealed
little or no mononuclear infiltration of the islets and no
degranulation or other destruction of the islet tissue when Tg/T2 lines
and clones were transferred (Fig. 2C). This result is in marked
contrast to transfers using either the BDC-2.5 clone (Fig. 2B) or diabetic spleen cells from the BDC-2.5 TCR Tg/NOD
mouse or BDC-2.5 TCR Tg/NOD.scid mouse, both of which lead
to rapid and extensive islet damage and hyperglycemia in
NOD.scid recipients.
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The results of experiments with the Tg/T2 lines in young
NOD.scid mice clearly indicated that these cells, although
diabetogenic, were working by a mechanism very different from that of
the Th1 T cell clones or diabetic T cells from the BDC-2.5 TCR Tg
mouse. The very efficient transfer of disease by the Tg/T2 line into
young NOD mice suggested that they might be working through recruitment
of host T cells. To test whether our transgenic Th2 lines and clones
could transfer diabetes to NOD.scid recipients in the
presence of "host cells," we performed cotransfers of the Tg/T2
line 2.5 Tg/T2-X with diabetic spleen cells. Our rationale for
performing this experiment was to determine whether the 2.5 Tg/T2-X
line, which is diabetogenic in the young NOD, would have an
accelerating effect on the diabetic spleen cells. As shown in Fig. 3a, cotransfer of the Tg/T2
line Tg/T2-X with diabetic spleen cells did indeed lead to an
acceleration of disease onset. After cotransfer, 50% of recipients
were diabetic by day 13, whereas when diabetic spleen cells were
transferred alone, only by day 18 were 50% of recipients diabetic.
This is a statistically significant acceleration with a p
value of 0.0005 by Log-Rank analysis. Cotransfers were also performed
using reduced numbers of diabetic spleen cells. As shown in Fig. 3, b and c, disease was accelerated to a greater
extent with transfer of two to four times fewer spleen cells. Fig. 4A shows islet infiltrate
after transfer of diabetic spleen cells plus 2.5 Tg/T2-X. Although
cotransferred mice became diabetic significantly earlier than mice that
received diabetic spleen cells alone, histological analysis did not
demonstrate any apparent difference between the two groups.
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The T cell clone BDC-6.3 is from our original panel of
islet-specific Th1 diabetogenic T cell clones (13), and
although it can cause diabetes in young NOD mice, it cannot induce
diabetes in the NOD.scid, even when the recipients are very
young. To determine whether a CD4+ T cell clone
alone could reconstitute an environment leading to disease induction by
a diabetogenic Tg/T2 line, we conducted cotransfer experiments with the
Tg/T2 line, 2.5 Tg/T2-X, and the BDC-6.3 clone in young
NOD.scid mice. As shown in Fig. 5, 7- to 12-day-old NOD.scid
mice transferred with either BDC-6.3 or 2.5 Tg/T2-X alone did not
become diabetic by day 28 postinjection. In contrast, seven of nine
mice that received both 2.5 Tg/T2-X and BDC-6.3 became diabetic by day
19. To demonstrate that this induction of disease was not merely a
result of greater cell numbers (2 x 107
cells in a cotransfer vs 1 x 107 cells when
clones were administered separately), we performed the cotransfer with
5 x 106 cells each of BDC-6.3 and 2.5
Tg/T2-X, and recipients (two of five) became diabetic by day 12 (data
not shown). It should be noted that the recipients in this group became
diabetic with the same kinetics as mice transferred with 2 x
107 total cells. We also found that transfer of
1 x 107 BDC-6.3 on day 0 and then injection
of 1 x 107 2.5 Tg/T2-X on day 7 led to
rapid diabetes onset in three of three recipients (data not shown).
Furthermore, we determined that animals receiving 1 x
107 cells of either BDC-6.3 or 2.5 Tg/T2-X alone
remained normoglycemic for greater than 60 days posttransfer (data not
shown). Histological analysis showed no significant infiltrate or
degranulation of islets in mice that received 2.5 Tg/T2-X alone (see
Fig. 2
C), although it is apparent that this clone gets into
the pancreas upon administration i.p. In some cases, incomplete
granulation was seen, as illustrated in Fig. 2A, but this is
typical in 3- to 6-wk-old NOD.scid mice in which some islets
are not completely granulated. Pancreatic sections from mice
transferred with BDC-6.3 alone indicated that this clone could also
migrate to the pancreas in that some islets showed mild to moderate
infiltrate and degranulation, although most were free of infiltrate and
completely granulated (Fig. 4B). In contrast, in the
cotransferred animals, there was complete degranulation of islets (Fig. 4C) accompanied by an extensive mononuclear infiltrate.
These data show that the Tg/T2 line 2.5 Tg/T2-X is not inert in the
NOD.scid and is in fact able to induce disease in the
presence of other effector T cells, even though it does not have the
ability to cause diabetes on its own.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite their apparent Th2 phenotype, these T cell lines caused rapid
diabetes onset in young NOD recipients. Surprisingly, however, transfer
of these cells did not lead to diabetes in young NOD.scid
mice, and theoretically NOD.scid mice less than 2 wk of age
should be the most susceptible recipients. This study is not the first attempt to obtain T cells with a Th2 phenotype from the 2.5 TCR Tg mouse. Katz et al. (33) previously reported success in producing IL-4-secreting T cells after culture of Tg T cells for 4 days under Th2-promoting conditions. These cultures were not diabetogenic in young NOD mice but also were not protective. A later publication by Pakala et al. (34) reported that TCR Tg T cells cultured under similar short-term conditions caused diabetes in adult NOD.scid mice but not in young NOD recipients. It is hard to evaluate the significance of this latter observation because diabetes is readily induced in young NOD.scid mice using Tg T cells subjected to no treatment whatsoever (our unpublished data). Furthermore, because a recent report by other investigators indicated that short-term Th2 cultures could not transfer diabetes to adult NOD.scid mice (36), the result of Pakala et al. may not be a consistent finding. Our original goal was to determine whether long-term Tg T cell lines with consistent cytokine production patterns would in fact prove to have protective properties. The establishment of long-term, stable Th2 T cell lines (especially from a TCR Tg mouse with a TCR from a Th1 T cell clone) is an important point because it is well documented in the literature that T cell cultures of less than 3 wk do not have a fixed cytokine production profile (37, 38, 39). As demonstrated previously by Murphy et al. (38), repeated restimulation of T cell cultures with specific Ag and Th1 and Th2 growth conditions can yield lines and clones with a "locked-in" phenotype, a feature we deemed critical to carrying out these studies. Furthermore, in the same report, it was found that low-level production of Th1 cytokines by short-term "Th2" lines was not detectable by ELISA and could only be detected by intracellular staining. Disease can be induced in NOD.scid recipients with as few as 1 x 105 spleen cells from a TCR Tg donor, so even a very small, undetectable population of Th1-like cells could account for the in vivo action of short-term Th2 cultures.
In contrast to the report of Pakala et al. (34) with short-term cultures, the work we have presented here has clearly demonstrated that cloned T cell lines with a Th2 phenotype can be diabetogenic in young NOD but not NOD.scid recipients. Furthermore, we have established that transfer effected by the Tg/T2 T cell lines takes place by a different mechanism than that of disease induced by Th1 T cell clones. Because the Tg/T2 lines readily cause diabetes in young NOD recipients but not in the lymphocyte-deficient NOD.scid, it was logical to speculate that they do so by recruiting host effector cells. This theory was supported by the experiment in which the Tg/T2 T cell line 2.5 Tg/T2-X was shown to accelerate disease transfer by diabetic spleen cells into young NOD.scid recipients. We felt that a more stringent test would be achieved if we could cotransfer the Tg/T2 cell line with a defined T cell clone incapable of causing disease in a NOD.scid recipient. The Th1 T cell clone BDC-6.3 is another CD4+ islet-specific clone from our original panel (13). This clone is very diabetogenic in young NOD mice but cannot by itself cause disease in the NOD.scid. Therefore, it was an excellent candidate for examining whether the Tg/T2 clone worked by "recruiting" other effector cells, and indeed, we found that cotransfer with BDC-6.3 led to rapid onset of diabetes in young NOD.scid recipients. The host cells in the NOD recipient might well include CD8 T cell effectors, but as in other work we have published (20), it is clear that a CD4 Th1 T cell clone is all that is needed for disease induction to take place.
One interpretation of our results with the Th2 T cell clones from the TCR Tg mouse is that these clones become "Th1-like" in vivo. We think this is unlikely. The parent BDC-2.5 clone causes disease in both young NOD and NOD.scid recipients, and at least in our hands, spleen cells from the 2.5 TCR Tg/NOD mouse transfer diabetes to young (and in some cases adult) NOD.scid mice. On the other hand, the Th2 T cell clones from the 2.5 TCR Tg mouse can induce disease only in young NOD mice (unless other cell populations are provided).
It is tempting to speculate that because the Th2 T cell clones from the
Tg mouse bear the receptor of what is obviously a very autoaggressive T
cell, they are diabetogenic because of their Ag specificity. Regardless
of the fact that they secrete Th2 cytokines, by virtue of their islet
reactivity they can initiate an inflammatory reaction in the pancreas
that attracts other Th1 effector T cells to the site. Because most of
the Tg/T2 T cell clones we have isolated fall into this category of
being diabetogenic in the young NOD, this is an attractive hypothesis.
However, we have isolated one Tg/T2 T cell clone that is not
diabetogenic in any circumstance we have tested, and this would suggest
that the Ag specificity may not be the only explanation. This clone,
like the others in the Tg/T2 panel, bears both the Vß4 and the V1
transgene from BDC-2.5. However, it is possible that it also bears some
endogenous TCR V that alters its functional reactivity.
There have been a variety of reports in the literature describing T
cell lines and clones with protective properties (reviewed in Ref.
25). There has also been much indirect evidence for a
protective role for T cells with the Th2 cytokine phenotype (i.e.,
IL-4/IL-10-secreting), including prevention of disease with recombinant
IL-4 and IL-10 therapy (29, 32) and transfer of protection
with spleen cells from NOD mice treated with IL-4 (30) or,
as demonstrated more recently, with T cells from mice immunized with
GAD-65 (40). Gallichan et al. (41) have also
recently shown that islet-reactive Th2 cells are responsible for the
protection from diabetes observed in transgenic NOD mice that bear the
IL-4 gene under the rat insulin promoter. The fact remains,
underscoring the importance of the work reported here, that there has
been no clear demonstration with defined, islet-reactive Th2 T cell
clones (i.e., IL-4+,
IL-10+, and IFN--) that
this type of T cell can delay, arrest, or prevent disease in the NOD
mouse. We were surprised to find that cloned T cell lines with a
reproducible pattern of Th2 cytokine production were not only not
protective but were in fact diabetogenic. Two major questions that
arise from our work are how a Th2 T cell clone can be diabetogenic and
what the requirements are for a Th2 T cell clone to be protective.
Whether the diabetogenic properties of our Tg/T2 T cells are due to the
specificity for Ag dictated by the BDC-2.5 TCR or to a lack of a
necessary factor (e.g., TGF-ß) is being addressed in studies
underway. Work in progress with another panel of Th2 T cell clones
derived from nontransgenic NOD mice and not yet demonstrated to be
diabetogenic may help to answer these questions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kathryn Haskins, Department of Immunology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B184, Denver, CO 80262. E-mail address:
3 Abbreviations used in this paper: NOD, nonobese diabetic; Tg, transgenic; A/F, aldehyde fuchsin; Tg/T2, Tg Th2.
Received for publication September 24, 1999. Accepted for publication January 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production is cytokine-dependent. Proc. Natl. Acad. Sci. USA 94:3189.This article has been cited by other articles:
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  T. Calzascia, M. Pellegrini, A. Lin, K. M. Garza, A. R. Elford, A. Shahinian, P. S. Ohashi, and T. W. Mak CD4 T cells, lymphopenia, and IL-7 in a multistep pathway to autoimmunity PNAS, February 26, 2008; 105(8): 2999 - 3004. [Abstract] [Full Text] [PDF]
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 J. Cantor and K. Haskins Recruitment and Activation of Macrophages by Pathogenic CD4 T Cells in Type 1 Diabetes: Evidence for Involvement of CCR8 and CCL1 J. Immunol., November 1, 2007; 179(9): 5760 - 5767. [Abstract] [Full Text] [PDF]
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J. Cantor and K. Haskins Effector Function of Diabetogenic CD4 Th1 T Cell Clones: A Central Role for TNF-{alpha} J. Immunol., December 1, 2005; 175(11): 7738 - 7745. [Abstract] [Full Text] [PDF]
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E. J. Woodward and J. W. Thomas Multiple Germline {kappa} Light Chains Generate Anti-Insulin B Cells in Nonobese Diabetic Mice J. Immunol., July 15, 2005; 175(2): 1073 - 1079. [Abstract] [Full Text] [PDF]
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S. S. Smith, T. Patterson, and M. E. Pauza Transgenic Ly-49A Inhibits Antigen-Driven T Cell Activation and Delays Diabetes J. Immunol., April 1, 2005; 174(7): 3897 - 3905. [Abstract] [Full Text] [PDF]
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M. A. Salam, K. Matin, N. Matsumoto, Y. Tsuha, N. Hanada, and H. Senpuku E2f1 Mutation Induces Early Onset of Diabetes and Sjogren's Syndrome in Nonobese Diabetic Mice J. Immunol., October 15, 2004; 173(8): 4908 - 4918. [Abstract] [Full Text] [PDF]
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H. Moriyama, N. Abiru, J. Paronen, K. Sikora, E. Liu, D. Miao, D. Devendra, J. Beilke, R. Gianani, R. G. Gill, et al. Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse PNAS, September 2, 2003; 100(18): 10376 - 10381. [Abstract] [Full Text] [PDF]
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C. Chen, W.-H. Lee, P. Yun, P. Snow, and C.-P. Liu Induction of Autoantigen-Specific Th2 and Tr1 Regulatory T Cells and Modulation of Autoimmune Diabetes J. Immunol., July 15, 2003; 171(2): 733 - 744. [Abstract] [Full Text] [PDF]
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D. V. Serreze, M. A. Pierce, C. M. Post, H. D. Chapman, H. Savage, R. T. Bronson, P. B. Rothman, and G. A. Cox Paralytic Autoimmune Myositis Develops in Nonobese Diabetic Mice Made Th1 Cytokine-Deficient by Expression of an IFN-{gamma} Receptor {beta}-Chain Transgene J. Immunol., March 1, 2003; 170(5): 2742 - 2749. [Abstract] [Full Text] [PDF]
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D. H. Wagner Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K. Haskins Expression of CD40 identifies a unique pathogenic T cell population in type 1 diabetes PNAS, March 19, 2002; 99(6): 3782 - 3787. [Abstract] [Full Text] [PDF]
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R. Tisch, B. Wang, M. A. Atkinson, D. V. Serreze, and R. Friedline A Glutamic Acid Decarboxylase 65-Specific Th2 Cell Clone Immunoregulates Autoimmune Diabetes in Nonobese Diabetic Mice J. Immunol., June 1, 2001; 166(11): 6925 - 6936. [Abstract] [Full Text] [PDF]
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Y. Chen, E. Rosloniec, M. I. Goral, M. Boothby, and J. Chen Redirection of T Cell Effector Function In Vivo and Enhanced Collagen-Induced Arthritis Mediated by an IL-2R{{beta}}/IL-4R{{alpha}} Chimeric Cytokine Receptor Transgene J. Immunol., March 15, 2001; 166(6): 4163 - 4169. [Abstract] [Full Text] [PDF]
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D. V. Serreze, H. D. Chapman, C. M. Post, E. A. Johnson, W. L. Suarez-Pinzon, and A. Rabinovitch Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation J. Immunol., January 15, 2001; 166(2): 1352 - 1359. [Abstract] [Full Text] [PDF]
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; n = 7)
or with 2 x 107 diabetic spleen cells plus 1 x
107 2.5 Tg/T2-X cells (•; n = 7).
b, NOD.scid mice received 1 x
107 diabetic spleen cells alone (
; n = 6),
with 1 x 107 of 2.5 Tg/T2-X (
;
n = 17), or with 1 x 107 of each
(