| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104;
Division of Nephrology, London Health Sciences Center, London, Ontaro, Canada; and
The Wistar Institute, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One important mechanism for the regulation of peripheral T cells is activation-induced clonal deletion, which occurs subsequent to a vigorous burst of proliferation in response to antigenic stimulation (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Optimal susceptibility of activated peripheral CD4 T cells to this Ag-driven activation-induced deletion process requires a threshold number of cell divisions (20). A failure to achieve this threshold could lead to the persistence of T cells normally subject to activation-induced regulation. In this work, the division history of activated NOD CD4 T cells was visualized, revealing that initiation of cell division is dependent upon cognate B cell costimulation. This reliance of CD4 T cell activation upon B cell-mediated costimulation in NOD mice is characterized by 1) a premature arrest of CD4 T cell division, which does not allow the threshold number of divisions required for optimal susceptibility to activation-induced deletion to be reached and 2) a resistance of CD4 T cells to TCR-mediated activation. This study demonstrates B cell-dependent aberrances in CD4 T cell activation that could contribute to dysregulated peripheral T cell tolerance in NOD mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NOD/LtJ, NOD.SWR (H-2q), NOD.Thy1.1 congenic, B6, B6.Thy1.1 congenic, and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 I-Aß-/- mice were purchased from Taconic Farms (Germantown, NY). NOD I-Aß+/- mice were generated by mating NOD I-Aß-/- mice (21) with wild-type NOD mice. All animals used in the described experiments were between 8 and 12 wk old and were housed in specific pathogen-free conditions at the University of Pennsylvania Medical Center. Purified anti-CD3 (2C11), purified anti-C28 (37.51), allophycocyanin-conjugated anti-CD4 (RM4-5), PE-conjugated anti-Vß3 (KJ25), PE-conjugated anti-Vß11 (RR3-15), and PE-conjugated anti-Thy1.1 (OX-7) were purchased from PharMingen (La Jolla, CA).
Lymphocyte enrichment protocol
Enrichment of T cells or APCs was accomplished by negative selection of splenocytes via magnetic-activated cell sorting (MACS; Miltenyi Biotec, Sunnyvale, CA). APCs were prepared by depleting T cells using anti-CD4, anti-CD8, and anti-Thy1.2 Abs directly conjugated to MACS beads (Miltenyi Biotec). Highly enriched T cells were prepared by depleting B lymphocytes using anti-B220 directly conjugated to MACS beads (Miltenyi Biotec) and biotinylated anti-I-Ag7 (10.3.6) plus streptavidin-MACS beads (Miltenyi Biotec). All depletion protocols yielded >95% efficiency in negative selection of the targeted population as determined by flow cytometry.
5-(and 6-)Carboxyfluorescein diacetate succinimidyl ester (CSFE) labeling of lymphocytes
Lymphocytes were labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (22). Briefly, a 5-mM stock solution of CFSE was prepared in DMSO. Lymphocytes isolated from spleens and lymph nodes of mice were resuspended at a concentration of 20–30 x 106 cells/ml in serum-free IMDM (Life Technologies, Gaithersburg, MD) at 37°C. An equal volume of a 1:250 dilution of the 5- mM CFSE stock in 37°C IMDM was added to the cell preparation. After a 5-min incubation period at 37°C, the excess CFSE was quenched by adding an equal volume of heat-inactivated FCS (HI-FCS). The CFSE-labeled cells were then washed once and resuspended at the desired concentration in IMDM containing 10% HI-FCS.
In vitro stimulation of CD4 T cells
CFSE-labeled cells were plated in 24-well plates at a density of 1 x 106 total cells/ml in media containing 10% HI-FCS, varying amounts of anti-CD3 (0–2 µg/ml), and 4 µg/ml anti-CD28 or staphylococcal enterotoxin A (SEA; Sigma, St. Louis, MO) at the designated concentration. For the plate-bound anti-CD3 stimulation assays, 24-well plates were coated with 0.5 ml of purified anti-CD3 in sterile PBS at 5 µg/ml overnight. After this incubation period, the plates were washed twice using sterile PBS to remove the excess unbound Ab. Highly enriched, CFSE-labeled T cells were then plated as described above. For the transwell experiments, 1 x 106 cells of the specified type were incubated in the top and bottom compartments separated by a mesh of 5-micron pore size. All cells were incubated for 65–70 h at 37°C in 7% CO2. After this incubation period, total output cell number was determined and the cultured cells were harvested and stained with allophycocyanin-conjugated anti-CD4 to allow the identification of CFSE-labeled CD4 T cells using FACScalibur (Becton Dickinson, Mountain View, CA). The total number and proportion of cells undergoing cell death following activation was determined by staining with Via-Probe (PharMingen), which labels dead cells with 7-amino actinomycin D (7-AAD). One hundred thousand total events were collected. Loss of CFSE intensity upon stimulation was used as a sensitive and reproducible measure of cell division.
Analysis of CD4 T cell division history
The fluorescent dye, CFSE, permits the direct visualization of lymphocyte division (22, 23, 24, 25, 26). When a CFSE-labeled population of lymphocytes divides, each round of division yields daughter cells that retain only half of the fluorescence intensity characteristic of the preceding parent population. The activated cell population is then analyzed by flow cytometry to determine the number of divisions and absolute number/proportion of daughter cells contained within each division peak. This information allows the direct calculation of the frequency and absolute number of the precursor cells that gave rise to the activated T cells using the total number of cells retrieved (23, 25). Briefly, the number of precursors giving rise to each daughter generation is determined by multiplying the total number of cells under each peak by a multiplication factor, 1/2n (where n is the division number). The division profiles determined at the end of the stimulation period can also be used to determine the total number of mitoses (23). This number is closely approximated by using the formula [N x (2n - 1)]/2n, where N is the total number of cells under each peak, n. The sum of the number of mitoses responsible for generating daughter cells under each division peak allows the determination of the total number of divisions that occurred during the stimulation period. Thus, by using CFSE to track the division history of a stimulated lymphocyte population, it is possible to obtain specific information regarding the frequency and absolute number of dividing cells as well as the number of divisions resulting from any degree of stimulation.
In vivo tracking and activation of CD4 T cells
CFSE-labeled splenocytes at a concentration of 2 x 106 cells/ml from NOD or C57BL/6 Thy1.1 congenic mice were stimulated with the designated amount of anti-CD3 and 4 µg/ml anti-CD28 for 65 h. Total activated CD4 T cells (25–30 x 106) were i.v. injected into wild-type NOD or C57BL/6 mice, respectively. For in vivo SEA-mediated activation and subsequent tracking of CD4 T cells, splenocytes and lymph node cells were isolated from NOD or C57BL/6 Thy1.1 congenic mice and CFSE labeled. These CFSE-labeled cells (30–40 x 106) were i.v. injected into wild-type NOD and C57BL/6 mice. After an overnight rest period to allow for homing of the transferred cells to the secondary lymphoid organs of the recipients, these mice were injected with 25–30 µg SEA in PBS. Kinetics of in vivo clearance of SEA-activated TCR Vß3/11-bearing CD4 T cells was determined by assessing the persistence of these cells relative to their maximal frequency achieved at 48–50 h following SEA injection, the time when the maximal proliferative burst is completed. At the designated time points following adoptive transfer of the described cells, spleens and lymph nodes were harvested from the recipient mice. Single cell suspensions were prepared and stained with anti-Thy1.1 PE-and allophycocyanin-conjugated anti-CD4 to allow for the identification of the transferred CD4 T cells using a FACScalibur. CD4+/Thy1.1+ events 2000–4000(2000–4000) were collected within a live lymphoid gate as determined by forward and side scatter.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have used CFSE to characterize the activation properties of NOD
CD4 T cells using the extent of cell division as a sensitive measure. A
combination of soluble mitogenic anti-CD3 and anti-CD28 mAb was
used to stimulate unfractionated CFSE-labeled splenocytes from NOD and
B6 mice. Using conditions providing maximal levels of activation by
anti-CD3 and anti-CD28 mAb, CD4 T cells from NOD mice exhibited
division arrest and failed to generate daughter cells in as advanced
division peaks as those achieved by their nonautoimmune B6 counterparts
(Fig. 1
A). Specifically,
comparison of the division profiles of NOD and B6 CD4 T cells indicated
that the majority (>80%) of activated NOD CD4 T cells resided in
divisions 1–4, whereas the majority (>70%) of activated B6 CD4 T
cells were present in divisions 4–7 (Fig. 1B). Assessment
of maximal anti-CD3/28-mediated CD4 T cell division by
nonautoimmune strain splenocyte isolates (B10, B6.g7, BALB/c, and C3H)
revealed an identical pattern of division to that of splenic B6 CD4 T
cells (data not shown).
|
C). In contrast, the starting pool of B6 CD4 T cells
directed the majority of its division effort to peaks 4–6 (Fig. 1C). Importantly, the diminished division potential of NOD
CD4 T cells was not imposed by the I-Ag7 MHC
haplotype as evidenced by identical division profiles of NOD.SWR
(H-2q) and wild-type NOD CD4 T cells (Fig. 1A). Moreover, B6.g7 CD4 T cells exhibited a maximal
division profile identical with that of wild-type B6 mice (data not
shown). NOD CD4 T cell division is highly sensitive to modulation of Ag ligand density
The diminished progression of division by maximally stimulated NOD
CD4 T cells as compared with their nonautoimmune strain counterparts
was evident at all concentrations of anti-CD3 tested (Fig. 2A). A dose-response curve was
generated using the calculated total number of mitoses occurring at
each concentration of anti-CD3 for both NOD and B6 CD4 T cells
(Fig. 2B). B6 CD4 T cells achieved their maximal division
state at 0.03 µg/ml anti-CD3, whereas NOD CD4 T cells achieved
their maximal division state at 1 µg/ml anti-CD3, an 
30-fold
higher concentration.
|
) and
I-A+/- B6 (data not shown) mice, whose APCs
express one-half the cell surface MHC class II density of wild-type
I-A+/+ counterparts, were used in SEA stimulation
assays. As demonstrated in Fig. 4A, B6 SEA-mediated CD4 T cell
activation is unaffected by a one-half reduction in MHC density at a
maximal stimulating SEA concentration of 15 µg/ml. At this maximal
level of stimulation, the ratio of activated CD4high to resting
CD4low T cells in B6 I-A+/+ and
I-A+/- cultures is nearly identical (a ratio of
2.5 in the I-A+/+ culture and 2.4 in the
I-A+/- culture; a 4% difference in the maximal
activation profiles). In fact, both the CFSE profile and CD4
distribution of SEA-activated B6 CD4 T cells from
I-A+/+ and I-A+/- mice
indicated that these cells had undergone a nearly identical
proliferation within a range of 1–15 µg/ml (Fig. 4A). In
contrast, NOD CD4 T cells stimulated with SEA in the context of the
one-half reduced I-A density of NOD I-A+/- APCs
exhibit a different activation profile when compared with their NOD
I-A+/+ counterparts (Fig. 4B). At the
maximum stimulating SEA concentration of 15 µg/ml, the ratio of
activated CD4high to resting CD4low T cells in the
I-A+/+ and I-A+/- cultures is different (a
ratio of 0.9 in the I-A+/+ culture and 0.45 in
the I-A+/- culture; a 50% difference in the
maximal division profile). Both the CFSE profile and CD4 distribution
of SEA-activated NOD CD4 T cells from I-A+/+ and
I-A+/- mice indicated that the activated T cells
in the I-A+/+ cultures achieved a more advanced
division state than the I-A+/- T cells at all
SEA concentrations from 1 to 15 µg/ml (Fig. 4B). It was
determined that the diminished proliferation of
I-A+/- NOD CD4 T cells was not due to a reduced
frequency of SEA-reactive CD4 T cells in the starting population (e.g.,
Vß11-bearing T cells) in the I-A+/- relative
to the I-A+/+ NOD mice (data not shown).
Additionally, CD4 T cells from both I-A+/+ and
I-A+/- NOD mice exhibited identical activation
characteristics when stimulated with soluble anti-CD3 in an
MHC-independent fashion (data not shown). This finding suggests that
the observed differences in the activation profiles of NOD CD4 T cells
stimulated in an MHC-dependent fashion, using SEA, are not due to
intrinsic differences between T cells from I-A+/+
vs I-A+/- mice. It is apparent that in addition
to a blunted division of CD4 T cells stimulated in the presence of
I-A+/- APC and SEA, fewer reactive cells were
stimulated, likely due to the limited availability of cell surface I-A.
This latter phenomenon has contributed to the overall diminished ratio
of CD4high to CD4low T cells following SEA stimulation
in the presence of I-A+/- APC compared with
I-A+/+ APC. In Fig. 4C we demonstrate
that cell surface CD4 expression level is an accurate measure of the
activation state of CD4 T cells. Specifically, the data indicate that
CD4high T cells in both NOD and C57BL/6 cultures stimulated
with SEA consist of divided CD4 T cells. Similarly, the CD4low
T cells in both NOD and C57BL/6 cultures consist of undivided CD4 T
cells. These patterns hold true at all concentrations of SEA from 1 to
15 µg/ml. This demonstration has allowed us to use CD4 expression
level as an accurate measure of the activation state of CD4 T cells in
the above discussion. In summary, consistent with their dose
responsiveness to anti-CD3/CD28, NOD CD4 T cells stimulated in the
described MHC-dependent assay are more sensitive to a one-half
reduction in the density of I-A ligand than their B6 counterparts. This
finding suggests that islet-reactive CD4 T cells in
I-A+/- NOD mice may not become sufficiently
activated to cause diabetes, a topic of current investigation in our
laboratory.
|
|
Recent work has suggested a dependence of efficient
activation-induced apoptosis on a threshold number of cell divisions
(20). Therefore, the failure of in vitro
anti-CD3-stimulated NOD CD4 T cells to achieve advanced divisions
prompted us to assess the efficiency of activation-induced death of
these cells. We determined that the majority of cell death, in both NOD
and B6 CD4 T cells, occurred after three divisions (Fig. 5A). Compared with maximally
stimulated B6 counterparts, NOD CD4 T cells maximally anti-CD3
stimulated failed to undergo cell death as vigorously (35 ± 8%
for NOD and 85 ± 4% for B6) (Fig. 5B). However, when
the number of cell divisions were equalized for both B6 and NOD CD4 T
cells by varying the concentration of anti-CD3, there was an
equivalent degree of activation-induced cell death by both B6 and NOD T
cells (43 ± 9% for B6 vs 35 ± 8% for NOD) (Fig. 5B). Importantly, quantification of the extent of
activation-induced death in the divided CD4+/CFSE
gate across the 0- to 2-µg/ml range of anti-CD3 stimulation
revealed a linear dependence of cell death upon the extent of cell
division in the case of both B6 and NOD CD4 T cells (Fig. 5, C and D, respectively). These data indicate that
the reduced susceptibility of NOD CD4 T cells to activation-induced
death may be a result of their inability to progress to advanced stages
of cell division.
|
We next determined whether the diminished capacity of activated
NOD CD4 T cells to progress through successive rounds of cell division
also prevents deletion in vivo. By using Thy1.1 congenic NOD and B6 T
cells, we were able to track CD4 T cells in vivo following adoptive
transfer into wild-type (Thy1.2) recipient mice. CD4 T cells from
Thy1.1 congenic NOD and B6 mice were CFSE labeled and maximally
stimulated in vitro with soluble anti-CD3 and anti-CD28 for
65 h. Cells were then adoptively transferred into wild-type NOD or
B6 mice. After a 10-day period, spleens and lymph nodes were isolated
from the recipient mice to determine the persistence of the maximally
activated Thy1.1+ CD4 T cells (Fig. 6, A and B). Very
few of the transferred population of maximally activated B6 CD4 T
cells, which were primarily in divisions 4–6 at the time of transfer,
were retrieved from the secondary lymphoid organs of the recipient mice
10 days later (Fig. 6B). In contrast, maximally divided NOD
CD4 T cells, which were distributed throughout divisions 1–5 at the
time of transfer, were persistent 10 days following adoptive transfer
into wild-type mice (Fig. 6A). To determine whether the in
vivo persistence of activated NOD CD4 T cells is brought about by
suboptimal cell division, we also stimulated Thy1.1 B6 CD4 T cells to
divide to a half-maximal level using anti-CD3 at 0.005 µg/ml in
conjunction with 4 µg/ml of anti-CD28. At this level of
stimulation, B6 CD4 T cells divided to the same extent as maximally
activated NOD CD4 T cells before in vivo transfer. After a 10-day
period, the half maximally activated B6 CD4 T cells persisted at a
level comparable to the maximally activated NOD CD4 T cells (Fig. 6C). The persisting cells were predominantly remaining
in divisions 1–3. Overall, these data demonstrate that a correlation
exists between the extent of cell division and susceptibility of
activated CD4 T cells to elimination from secondary lymphoid organs
in vivo.
|
A). Fig. 7B is a
time course analysis of the in vivo clearance of a SEA-activated
Vß3/Vß11-bearing CD4 T cell marker population in NOD and B6 mice.
Approximately 90% of the activated B6 CD4 T cell population seen at
48 h were deleted in vivo by 200 h following SEA injection.
In contrast, only 60% of the activated NOD CD4 T cell population seen
at 48 h were subject to in vivo elimination by 200 h
following SEA injection. We had previously observed that in vitro
SEA-mediated activation of NOD CD4 T cells, similar to
anti-CD3-mediated stimulation, resulted in a premature termination
of cell division relative to B6 counterparts (Fig. 4). Thus, the
long-term in vivo persistence of SEA-activated NOD CD4 T cells is
associated with a blunted cell division profile.
|
Efficient T cell activation and, in particular,
anti-CD3-mediated activation requires the presence of competent
APCs as a source of costimulation (27). Given the impaired
activation of NOD CD4 T cells, we sought to determine whether these
characteristics are T cell intrinsic or are imposed by aberrant NOD APC
function. Thus, we assessed the ability of both the B cell and non-B
cell APC compartments for delivery of the costimulation necessary to
drive effective CD4 T cell activation. In the absence of B cells, NOD
CD4 T cells failed to initiate division even upon maximal stimulation
with soluble anti-CD3/28 (Fig. 8). In
marked contrast, B6 CD4 T cells remained capable of undergoing
successive rounds of division in the absence of B cells. The profound
unresponsiveness of NOD CD4 T cells in the absence of B lymphocytes was
irreversible even upon addition of exogenous rIL-2 (Fig. 8). This
latter finding suggests that the proliferative unresponsiveness of NOD
CD4 T cells in the absence of B cells does not fit the conventional
definition of T cell "anergy" (28). Furthermore, using
plate-bound anti-CD3, an APC-independent T cell functional assay,
we stimulated highly enriched NOD and B6 T cells in the complete
absence of B and non-B cell APCs for 70 h (Fig. 8B). In
this plate-bound anti-CD3 assay, in contrast to APC-dependent
stimulation with soluble anti-CD3/28, NOD and B6 CD4 T cells
exhibited a comparable division profile. These data indicate that NOD
CD4 T cells do not exhibit an intrinsic defect in their ability to
become activated; rather, the observed NOD CD4 T cell activation defect
in response to soluble anti-CD3/28 resides in the inability of the
non-B cell NOD APC compartment to provide optimal costimulatory
signals. Given this aberrance in NOD APC function, we hypothesized that
the suboptimal division profile of activated NOD CD4 T cells in
response to soluble anti-CD3/28 is due to a non-B cell APC deficit,
which imposes a reliance on B cell-mediated costimulation for CD4 T
cell activation. Therefore, purified NOD CD4 T cells were maximally
stimulated with soluble anti-CD3/CD28 in the presence of
nonautoimmune B6 APCs. In the presence of these nonautoimmune strain
APCs, NOD CD4 T cells divided to a nearly identical level as their
maximally stimulated B6 counterparts (Fig. 9A). This potentiated division
of NOD CD4 T cells occurred when these cells were stimulated in the
presence of either I-A+/+ or
I-A-/- B6 APCs, indicating that the observed
enhancement of cell division was not due to an alloresponse directed
toward the I-Ab MHC expressed by the B6 APCs. In
fact, B6 strain APCs delivered an intrinsically different,
MHC-independent, costimulatory signal to CD4 T cells, which mediated an
enhanced proliferation of NOD CD4 T cells as compared with that seen
upon stimulation in the presence of NOD APCs. This normalization of NOD
CD4 T cell division upon receipt of costimulation via B6 APCs also
brought about an enhanced degree of activation-induced cell death (Fig. 9B). Furthermore, the linear relationship that existed
between cell division and activation-induced death (Fig. 5, C and D) was preserved when NOD CD4 T cells were
stimulated in the presence of B6 APCs (Fig. 9B).
|
|
In vitro stimulation of T cells using soluble anti-CD3,
despite being MHC independent, requires the presence of costimulatory
signals from Fc receptor-bearing APCs (29). Data presented
above indicate that, in contrast to B6 counterparts, NOD CD4 T cells
require B cells for the initiation of anti-CD3-mediated activation.
We hypothesized that the costimulation provided to NOD CD4 T cells by B
lymphocytes is delivered via a cognate interaction between B and T
cells. CFSE-labeled, fractionated NOD T and B cells were plated in
separate compartments of a transwell system while stimulating the T
cells with anti-CD3 and anti-CD28 for 65 h. NOD CD4 T
cells failed to initiate division despite the presence of B cells in
the transwell system, indicating a requirement for cognate T/B cell
interactions (Fig. 10A).
Furthermore, the soluble products of unfractionated NOD splenocyte
activation with anti-CD3/CD28 did not induce fractionated NOD CD4 T
cells to divide in the transwell studies (Fig. 10B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The resistance of NOD CD4 T cells to the initiation of cell division may render activation on a per cell basis ineffective when a large frequency of T cells compete for the same antigenic ligand(s). It could be argued that competition among CD4 T cells for an antigenic niche would decrease the stimulus received on a per cell basis. Such competition could lead to suboptimal activation of NOD CD4 T cells given their diminished dose responsiveness compared with nonautoimmune counterparts. This latter prediction may be the explanation for an intriguing finding recently described by the Benoist and Mathis laboratory in the BDC2.5 TCR transgene (Tg) model of autoimmune diabetes (6). The T cell compartment of BDC2.5 Tg mice contains a high frequency of potentially diabetogenic CD4 T cells with monoclonal specificity. When the BDC2.5 Tg was fully backcrossed onto the NOD genetic background, diabetogenic T cells were not sufficiently activated to cause diabetes despite impressive homing of Tg T cells to the islets. However, in B6 mice congenic for H-2g7, the presence of the BDC2.5 TCR Tg readily induces diabetes, pointing to the efficient activation of the diabetogenic Tg T cells on the B6 genetic background. This dichotomy may be explained by our finding that NOD CD4 T cells require a dramatically higher stimulus for activation than their B6 counterparts. Indeed, on the NOD genetic background, it is competition between the supraphysiologic number of Tg-encoded BDC2.5 T cells which may be impeding sufficient activation of individual cells. In contrast, BDC2.5 T cells on the B6 genetic background, due to their significantly lower activation threshold, are not subject to the same competitive force and thereby receive enough stimulation at the single cell level to achieve their full pathogenic potential.
Despite their resistance to activation upon TCR complex ligation, NOD CD4 T cells are capable of dividing to generate activated progeny cells. However, even upon maximal stimulation, NOD CD4 T cell division is prematurely aborted such that the majority of activated progeny cells are distributed in the first three division peaks. This phenotype was also observed in activated CD4 T cells from NZB/NZW F1 and MRL mice, which are prone to spontaneous autoimmunity (data not shown). In contrast, maximally stimulated CD4 T cells from nonautoimmune mice consistently progressed beyond the third cell division. The inability of CD4 T cells to efficiently progress to advanced cell divisions upon stimulation through the TCR complex may be a general characteristic of strains susceptible to spontaneous autoimmunity.
Several recent studies have suggested that a threshold number of cell divisions is required for certain differentiated T cell functions to optimally progress. One such division-dependent process is the commitment of activated CD4 T cells to an IL-4-secreting phenotype that requires a minimum of three cell divisions (24). In light of this recent report, our finding that NOD CD4 T cells prematurely terminate cell division is a potential explanation for the profound inability of activated peripheral T cells to produce IL-4 (32, 33, 34), a deficit that is a shared characteristic of human type I diabetics and NOD mice (35). Indeed, an inefficiency in the differentiation of IL-4-producing "regulatory" Th2 cells has been suggested to allow the development of autoimmune diabetes in NOD mice (3). Another important division-dependent process is the susceptibility of CD4 T cells to activation-induced death. It was recently shown that apoptosis of superantigen-activated T cells occurs most efficiently following three cell divisions (20). Thus, we predicted that the inability of NOD CD4 T cells to efficiently progress to advanced cell divisions may lead to the abnormal persistence of activated T cells. Assessment of the extent of activation-induced death revealed a linear dependence upon the degree of cell division in the case of both activated NOD and B6 CD4 T cells. Our results directly demonstrate that the elimination of CD4 T cells following in vivo activation is a function of their division state. We conclude that long-term persistence of activated NOD CD4 T cells occurs as a consequence of their inability to exceed the division threshold necessary for efficient in vivo activation-induced deletion. In fact, a study by the Holmberg laboratory has indicated that NOD mice mount a significantly more vigorous and prolonged secondary immune response to a foreign immunogen, and that this phenotype correlates with the resistance of NOD T cells to death (36). It is possible that the persistence of suboptimally activated (and, therefore, nondiabetogenic) islet-reactive T cells in the benign insulitis lesion seen in younger NOD mice (4–12 wk of age) may be the result of a persistent T cell response to islets, much like that seen against foreign immunogens. The factor(s) that allow sufficient activation of the anti-islet T cells in the benign insulitis of younger NOD mice to a diabetogenic phenotype in older NOD mice remain to be elucidated.
Work from the Fathman group indicates that peripheral T cell tolerance to ubiquitous self-Ags can be readily broken in NOD mice. Following immunization with autoantigen, a promiscuous and persistent state of Ag-specific CD4 T cell activation, termed "autoproliferation," occurred (37). A seemingly contradictory body of work from Delovitch and colleagues documented a marked degree of unresponsiveness by purified NOD CD4 T cells upon TCR complex ligation (34, 38, 39, 40). This generalized defect in NOD T cell activation was interpreted to be indicative of a global state of T cell "anergy." The apparent contradiction between CD4 T cell anergy and promiscuous "autoproliferation" is reconcilable by our results demonstrating that the reduced division capacity of activated NOD CD4 T cells (i.e., anergy) directly leads to the long-term in vivo persistence of these cells, which may account for the "autoproliferative" phenotype.
In light of the observed aberrances in the activation characteristics
of NOD CD4 T cells, we questioned whether these defects are T cell
intrinsic or are imposed by the NOD APC compartment. The presented data
indicate that NOD CD4 T cells are not intrinsically defective in their
ability to become activated. Rather, it is the non-B cell NOD APCs that
are unable to provide the costimulation necessary to drive CD4 T cell
division, leading to the observed activation defects in APC-dependent T
cell functional assays. Indeed, this abnormality in non-B cell NOD APC
function is consistent with several studies characterizing a defect in
the development and function of these APCs (41, 42, 43, 44). Our
data indicate that cognate B cell costimulation is required for driving
soluble anti-CD3 mediated cell division. Thus, we suggest that B
cells may be the most functional APC compartment in the NOD mouse.
Indeed, it was recently demonstrated that the progression of diabetes
in wild-type NOD mice requires the presence of B lymphocytes, which act
as the requisite APCs in the activation of
I-Ag7-restricted diabetogenic T cells
(45, 46, 47, 48, 49, 50). Moreover, the Sarvetnick laboratory has recently
demonstrated that diabetogenesis in NOD mice is dependent upon
polarized B cell-mediated Ag presentation to islet-reactive T cells
(51). It was shown that when the NOD immune system is
modulated to preferentially use macrophages as APCs, the mice are
protected from autoimmune diabetes. Furthermore, data from the Flavell
group indicate that a dramatic increase in the number of B lymphocytes
in the insulitic lesion is correlated with early onset of autoimmune
diabetes in receptor interacting protein-TNF-
Tg mice
(52). These finding are consistent with our contention
that B lymphocytes are the dominant APCs in wild-type NOD mice and are
required for the activation of diabetogenic T cells. However, B
cell-mediated costimulation in NOD mice falls short of mediating the
degree of activation required for regulation of autoreactive T cells
throughout the ontogeny of the T cell compartment.
Collectively, the presented data suggest that the susceptibility of NOD mice to autoimmune diabetes may involve 1) impaired T cell activation resulting from suboptimal costimulation by NOD APCs, which leads to inefficient activation-induced regulation of islet-reactive T cells and 2) a dominance of B cell costimulation in the activation of diabetogenic T cell responses in wild-type NOD mice.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ali Naji, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104.
3 Abbreviations used in this paper: NOD, nonobese diabetic; CFSE, 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester; SEA, staphylococcal enterotoxin A; Tg, transgene; MACS, magnetic-activated cell sorting; HI-FCS, heat-inactivated FCS; 7-AAD, 7-amino actinomycin D.
Received for publication March 9, 2000. Accepted for publication July 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
define distinct epitopes, one of which may interact with CD4 during T cell activation. J. Immunol. 142:4169.[Abstract]
24JQ T cells in type 1 diabetes. Nature 391:177.[Medline]
in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens. Immunity 9:733.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. L. Kendall, D. J. Moore, C. Hulbert, K. L. Hoek, W. N. Khan, and J. W. Thomas Reduced Diabetes in btk-Deficient Nonobese Diabetic Mice and Restoration of Diabetes with Provision of an Anti-Insulin IgH Chain Transgene J. Immunol., November 15, 2009; 183(10): 6403 - 6412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Smith and T. F. Tedder Targeting B-cells Mitigates Autoimmune Diabetes in NOD Mice: What Is Plan B? Diabetes, July 1, 2009; 58(7): 1479 - 1481. [Full Text] [PDF]
|
|
|
|
|
|
G. Zekavat, S. Y. Rostami, A. Badkerhanian, R. F. Parsons, B. Koeberlein, M. Yu, C. D. Ward, T.-S. Migone, L. Yu, G. S. Eisenbarth, et al. In Vivo BLyS/BAFF Neutralization Ameliorates Islet-Directed Autoimmunity in Nonobese Diabetic Mice J. Immunol., December 1, 2008; 181(11): 8133 - 8144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Brodie, M. Wallberg, P. Santamaria, F. S. Wong, and E. A. Green B-Cells Promote Intra-Islet CD8+ Cytotoxic T-Cell Survival to Enhance Type 1 Diabetes Diabetes, April 1, 2008; 57(4): 909 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bour-Jordan, B. L. Salomon, H. L. Thompson, R. Santos, A. K. Abbas, and J. A. Bluestone Constitutive Expression of B7-1 on B Cells Uncovers Autoimmunity toward the B Cell Compartment in the Nonobese Diabetic Mouse J. Immunol., July 15, 2007; 179(2): 1004 - 1012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. Quinn III, N. Noorchashm, J. E. Crowley, A. J. Reed, H. Noorchashm, A. Naji, and M. P. Cancro Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse. J. Immunol., June 15, 2006; 176(12): 7159 - 7164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Moore, H. Noorchashm, T. H. Lin, S. A. Greeley, and A. Naji NOD B-cells Are Insufficient to Incite T-Cell-Mediated Anti-islet Autoimmunity Diabetes, July 1, 2005; 54(7): 2019 - 2025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sblattero, F. Maurano, G. Mazzarella, M. Rossi, S. Auricchio, F. Florian, F. Ziberna, A. Tommasini, T. Not, A. Ventura, et al. Characterization of the Anti-Tissue Transglutaminase Antibody Response in Nonobese Diabetic Mice J. Immunol., May 1, 2005; 174(9): 5830 - 5836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Acevedo-Suarez, C. Hulbert, E. J. Woodward, and J. W. Thomas Uncoupling of Anergy from Developmental Arrest in Anti-Insulin B Cells Supports the Development of Autoimmune Diabetes J. Immunol., January 15, 2005; 174(2): 827 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Vence, C. Benoist, and D. Mathis Fas Deficiency Prevents Type 1 Diabetes by Inducing Hyporesponsiveness in Islet {beta}-Cell-Reactive T-Cells Diabetes, November 1, 2004; 53(11): 2797 - 2803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hussain, K. V. Salojin, and T. L. Delovitch Hyperresponsiveness, Resistance to B-Cell Receptor--Dependent Activation-Induced Cell Death, and Accumulation of Hyperactivated B-Cells in Islets Is Associated With the Onset of Insulitis but not Type 1 Diabetes Diabetes, August 1, 2004; 53(8): 2003 - 2011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Silveira, J. Dombrowsky, E. Johnson, H. D. Chapman, D. Nemazee, and D. V. Serreze B Cell Selection Defects Underlie the Development of Diabetogenic APCs in Nonobese Diabetic Mice J. Immunol., April 15, 2004; 172(8): 5086 - 5094. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Makhlouf, A. Yamada, T. Ito, R. Abdi, M. J. I. Ansari, C. Q. Khuong, H. J. Winn, H. Auchincloss Jr., and M. H. Sayegh Allorecognition and Effector Pathways of Islet Allograft Rejection in Normal versus Nonobese Diabetic Mice J. Am. Soc. Nephrol., August 1, 2003; 14(8): 2168 - 2175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M.-L. Bergman, N. Duarte, S. Campino, M. Lundholm, V. Motta, K. Lejon, C. Penha-Goncalves, and D. Holmberg Diabetes Protection and Restoration of Thymocyte Apoptosis in NOD Idd6 Congenic Strains Diabetes, July 1, 2003; 52(7): 1677 - 1682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Pearson, T. G. Markees, D. V. Serreze, M. A. Pierce, M. P. Marron, L. S. Wicker, L. B. Peterson, L. D. Shultz, J. P. Mordes, A. A. Rossini, et al. Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 185 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sen, S. Bhattacharyya, M. Wallet, C. P. Wong, B. Poligone, M. Sen, A. S. Baldwin Jr., and R. Tisch NF-{kappa}B Hyperactivation Has Differential Effects on the APC Function of Nonobese Diabetic Mouse Macrophages J. Immunol., February 15, 2003; 170(4): 1770 - 1780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. P. L. Chiu, A. M. Jevnikar, and J. S. Danska Genetic Control of T and B Lymphocyte Activation in Nonobese Diabetic Mice J. Immunol., December 15, 2001; 167(12): 7169 - 7179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. W. Greeley, D. J. Moore, H. Noorchashm, L. E. Noto, S. Y. Rostami, A. Schlachterman, H. K. Song, B. Koeberlein, C. F. Barker, and A. Naji Impaired Activation of Islet-Reactive CD4 T Cells in Pancreatic Lymph Nodes of B Cell-Deficient Nonobese Diabetic Mice J. Immunol., October 15, 2001; 167(8): 4351 - 4357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Johnson, P. Silveira, H. D. Chapman, E. H. Leiter, and D. V. Serreze Inhibition of Autoimmune Diabetes in Nonobese Diabetic Mice by Transgenic Restoration of H2-E MHC Class II Expression: Additive, But Unequal, Involvement of Multiple APC Subtypes J. Immunol., August 15, 2001; 167(4): 2404 - 2410. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
; B6, 
).
C, Determination of the contribution of maximally
stimulated CD4 T cell precursors to the activated progeny cells in each
division peak. The bars designate the average of the dots each
representing data obtained from an individual mouse (NOD, 
). The number of mitoses occurring at each concentration of
anti-CD3 relative to the number of mitoses achieved upon maximal
activation comprises the abscissa of the dose response plot. The dotted
lines indicate the concentration of anti-CD3 at which the maximal
division of B6 and NOD CD4 T cells occurs.
) and B6 (
) mice. By 120 h following SEA injection, 
