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*
Section of Immunobiology, Yale University School of Medicine, and
Howard Hughes Medical Institute, New Haven, CT 06520; and
Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109
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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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In diabetic NOD mice, pancreatic islets are infiltrated by CD4+ T cells and at lower levels by CD8+ T cells, B lymphocytes, monocytes, macrophages, dendritic cells, and NK cells (1, 4). Studies with athymic NOD nude mice (5, 6) have clearly documented the importance of T lymphocytes in the onset of diabetes in NOD mice; however, the precise role of T cell subsets remains unclear. Insulitis and ß-cell destruction are clearly mediated by these invading T cells (7). The relative importance of CD4+ and CD8+ T cells in the pathogenesis of diabetes has been widely discussed (6, 8, 9, 10, 11, 12, 13, 14, 15). Some authors have proposed that CD8+ T cells are only required for effective priming and expansion of autoreactive CD4+ T cells (12), while others have shown that cloned autoaggressive CD8+ T cells are able to cause diabetes by themselves (13). It is critical, then, to determine which population triggers the diabetogenic process to design effective therapeutic approaches.
CIITA (16) is a non-DNA-binding transcription factor that is required
for the constitutive and IFN-
-induced expression of conventional MHC
class II molecules. CIITA is also involved in the expression of
molecules related to Ag presentation, such as the invariant chain and
H2-M (16, 17, 18, 19, 20, 21). There is evidence showing that CIITA is also required
for IFN--inducible expression of MHC class I in murine and human
cell lines (22, 23). A recent report, however, showed that macrophages
elicited from CIITA-deficient mice are able to up-regulate MHC class I
upon IFN- stimulation, suggesting that this mechanism may not be
critical in vivo (24). Mice deficient in CIITA (CIITA KO) are deficient
in MHC class II expression and show an impairment in the expression of
invariant chain and H2-M molecules (18, 24). CIITA KO mice have
markedly decreased numbers of peripheral CD4+ T cells due
to a defect in thymic positive selection because of the relative
absence of MHC class II molecules (25, 26, 27). The repertoire of
CD8+ T cells remains intact (18).
To elucidate the role of MHC class II-restricted CD4+ T cells in the pathogenesis of IDDM we have studied the development of diabetes in CIITA-deficient mice on the NOD background. CIITA KO/NOD mice, then, were superficially similar to MHC class II KO/NOD mice. However, in NOD/MHC class II KO mice both diabetes and pancreatic infiltration were abrogated (28), and the same phenotype was exhibited by the heterozygous littermates (28). Since the MHC class II genes are located on chromosome 17, closely linked to MHC class I, the MHC class I and other MHC alleles linked to the disrupted ß-chain in the MHC class II gene (originally gene targeted in 129/Sv embryonic stem cells and backcrossed onto the C57/BL6 background), also derive from the 129/Sv-C57/BL6 strains whose haplotype for MHC class I is H-2Kb and H-2Db. Due to the tight linkage between both MHC class I and class II, backcrossing onto NOD does not substitute the MHC class I diabetes-resistant alleles (i.e., H-2Kb) for the NOD-susceptible alleles (i.e., H-2Kd). Thus, effects observed in the MHC class II KO mice cannot readily be interpreted (28). The CIITA gene, unlike MHC class I and II genes, is not located on chromosome 17, so NOD class I molecules (H-2Kd and H-2Db) are expressed normally instead of being replaced by MHC class I genes of the MHC class II KO mice originally derived from the 129/Sv strain. NOD/CIITA KO mice, then, provide a cleaner model to study the role of CD4 T cells in the pathogenesis of IDDM. In contrast to MHC class II KO mice, NOD/CIITA KO mice exhibit pancreatic infiltration, although seldom insulitis, and they do not develop diabetes. A normal CD4 T cell repertoire is required for severe insulitis and diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CIITA KO mice were previously generated in our laboratory by gene-targeted disruption, and homozygosity for the mutation was obtained by intercrossing on the C57BL/6 background (18). The CIITA KO mice were backcrossed five times onto the NOD background, selecting from the third generation those mice homozygous for the highest number of NOD Idd alleles. In the fourth generation one female was already homozygous for all 15 Idd NOD alleles. This female was backcrossed once again onto the NOD background, and the progeny was intercrossed to provide experimental mice. Screening for the CIITA mutation was performed using Southern analysis as previously described (18). Unless otherwise stated all NOD/CIITA mice used correspond to the fifth backcross. We have further backcrossed onto the NOD background up to 10 generations (N11) for some of the experiments performed.
Genotyping for the Idd loci was performed by using PCR primers specific for the different loci that are polymorphic for the 129/Sv, C57BL/6, and NOD strains. The primers used were: Idd1 (H-2g7; for Idd1 screening two sets of PCR primers were used, a and b: a, D17 Mit34; b, 5'-TGT CTT TTC TGT CAC CCT AGA ACA-3', 3'-TAC ACC TCG TAG GGT CGG ACT TCT C-5'; the PCR product obtained by b primers (282 bp) was digested using MspI; the NOD allele is resistant to cleavage (29)); Idd2, D9 Mit25; Idd3, D3Nds36; Idd4, D11 Mit 115, D11 Mit320; Idd5, D1 Mit24, D1 Mit26; Idd6, D6 Mit52; Idd7, D7 Mit20; Idd8, D14 Mit11; Idd9, D4 Mit59; Idd10, D3 Mit103; Idd11, D4 Mit202; Idd12, D14Nds3; Idd13, D2 Mit395; Idd14, D13 Mit61; and Idd15, D5 Mit48. The PCR products were run in 4% agarose gels.
Assessment of diabetes and immunohistochemical analysis
Female NOD/CIITA N6 (fifth backcross) KO (-/-) or their CIITA-sufficient (+/+ or +/-) littermates were monitored weekly for the development of glycosuria with Diastix (Ames, Elkhart, IN), starting at 7 wk of age. Diabetes was confirmed by measuring glucose levels in the blood. Glycemia was measured using One Touch test strips (Lifescan, Johnson & Johnson, Palmitas, CA), and values over 250 mg/dl (>13.9 mM) were considered positive for diabetes. Pancreata were either fixed in 10% buffered formalin or processed for immunohistochemistry by fixation in paraformaldehyde-lysine-periodate buffer. In the former case the tissue was embedded in paraffin, sectioned, and stained with hematoxylin-eosin to assess the presence of mononuclear infiltrate in the pancreatic islets (insulitis). For immunohistochemistry, after 16 h in paraformaldehyde-lysine-periodate buffer at 4°C the tissue was incubated in increasing concentrations of sucrose dissolved in phosphate buffer (10, 20, and 30%), embedded in Tissue-Tek OCT (Miles, Elkhart, IL), and frozen in 2-methylbutane (isopentane). Five- to seven-micron-thick frozen sections were taken and stained for insulin and glucagon (alkaline-phosphatase kit, BioGenex, San Ramon, CA); for mouse CD4, CD8, B220, and Gr-1 (PharMingen, San Diego, CA); and for F4/80 (Serotec, Raleigh, NC). The sections were incubated in the presence of alkaline-phosphatase-conjugated streptavidin and subsequently in the presence of the substrate HistoMark Red (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for the development of color.
To assess the degree of pancreatic infiltration, sections were taken, and islets were counted. To estimate the percentage of peri-infiltrated or infiltrated islets, at least 10 islets were counted in each field, and at least two different fields were observed. In each estimation no fewer than 3 mice were included. For quantitative analysis of the infiltrates similar criteria were followed.
Isolation of PBL
Mice were bled through the retro-orbital vein using heparinized tubes. Blood mononuclear cells were isolated by centrifuging total blood in a Ficoll gradient (Organon Teknika, Durham, NC) following the manufacturer’s instructions.
Adoptive transfer experiments
Recipient mice (older than 4 wk) were irradiated using 725 rad from a cesium source 24–48 h before adoptive transfer. The source of the transferred cells varied in each experiment. If they were originally isolated from the spleen, total splenocytes were isolated in sterile conditions by physical disruption of the spleen using frosted glass slides, then RBC were lysed by hypotonic shock with distilled water. For adoptive transfer in which the CD4 or CD8 T cell subsets were purified from splenocytes, IsoCell (CD4 or CD8) isolation columns were used (Pierce, Rockford, IL), following the manufacturer’s instructions. The purity of the fraction obtained in the desired T cell population was >90%. TGNFC8 cloned CD8 T cells were activated in vitro as previously described (13) and adoptively transferred subsequently. In all adoptive transfer experiments cells were transferred in 200 µl of physiological saline (0.9% NaCl) by i.v. injection.
Flow cytometric analysis of spleen cells
Splenocytes were isolated as described above and stained following the standard procedure in PBS supplemented with 1% FBS (for directly conjugated Abs) or 2% BSA in PBS (for biotinylated Abs). The Abs used for flow cytometric analysis (CD4, CD8, CD3, B220, CD23, CD40, CD62L, CD69, B7-1, and B7-2) were directly conjugated to one of the following fluorochromes: FITC, phycoerythrin, or Cychrome (PharMingen). Only the Abs used for MHC class II detection (the clones 10.2.16 (American Type Culture Collection, Manassas, VA) and AMS-32.1 (PharMingen)) were biotinylated. Incubation with directly conjugated Abs or biotinylated Abs was conducted at 4°C for 30 min. Biotinylated Abs were detected using streptavidin-FITC, -phycoerythrin, or -Cy-Chrome. After the staining process the cells were analyzed on FACS IV (Becton Dickinson Immunocytometry Systems, Mountain View, CA). For FACS cell sorting, Ab solutions were filtered (0.2 µm pore size), and the staining process was conducted under sterile conditions.
Depletion of CD4 T cells with magnetic beads
Total spleen cells were isolated as described above and
incubated at 4°C for 30 min in the presence of rat anti-mouse CD4
Ab (GK1.5, American Type Culture Collection); they were then incubated
in the presence of magnetic beads conjugated to goat anti-mouse IgG
(BioMagsé, PerSeptive Biosystems, Framingham, MA) for two rounds
to deplete CD4-positive cells. As a negative control of depletion half
the initial spleen cells were incubated only in the presence of the
magnetic beads (nondepleted group). The degree of depletion was
ascertained by staining for the presence of CD4-, CD8-, and
B220-positive cells in the spleen fractions before and after depletion.
The depletion of CD4 cells achieved following this protocol was
remarkably satisfactory; from 96–99% of CD4 cells were eliminated
(see Fig. 3
D, a and b).
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On day 0 CD8 T cells from 3- or 18-wk-old NOD/CIITA KO or wt
males were FACS sorted using anti-mouse Cy-Chrome-conjugated CD8
Ab. Sorted cells were cultured in Clicks (Bruff’s) medium supplemented
with 10% FBS and seeded in U-bottom 96-well plates at 105
cells/well in the presence or the absence of 2 x 105
irradiated (3000 rad from a cesium source) total spleen cells from
nondiabetic 4-wk-old NOD females as APCs. Con A was used for in vitro
stimulation at 2.5 µg/ml on day 0. On day 1 the activation state of
the cells was assessed by staining for CD69 and CD62L expression on the
cell surface. On day 4 cells were restimulated with Con A (2.5 µg/ml;
Boehringer Mannheim, Indianapolis, IN), and supernatants were collected
on day 5 to assess cytokine production (IFN- and IL-4) by ELISA (the
unconjugated capture and biotinylated detection Abs were purchased from
PharMingen). On day 3 wells were pulsed with 1 µCi of
[3H]thymidine (New England Nuclear, Boston, MA) and were
harvested 16–24 h later.
In vitro activation of B cells
On day 0 B220+ cells were sorted and then cultured in Clicks (Bruff’s) medium supplemented with 5% FBS at 105 cells/well in U-bottom 96-well plates. LPS alone (10 µg/ml; Salmonella typhimurium; Sigma, St. Louis, MO) or LPS and 20 U/ml of recombinant mouse IL-4 (DNAX, Palo Alto, CA) were added to activate B cells. On day 1 or 2 cells were harvested to assess the expression of the activation markers on the cell surface by FACS analysis (CD23, B7.1, B7.2, and CD40 (day 1); MHC class II (day 2)). On day 2 cells were pulsed with 1 µCi of [3H]thymidine and were harvested after 16–24 h. For the detection of Ig production supernatants were collected on day 7 and assayed for the presence of IgM, IgG1, IgG2a, and IgG2b by ELISA (the unconjugated capture and biotinylated detection Abs were purchased from Southern Biotechnology Associates, Birmingham, AL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CIITA KO mice were generated by targeted disruption of the CIITA
locus and were backcrossed to the C57/BL6 background (18). We
backcrossed these mice for five generations onto the NOD background,
selecting for those mice homozygous for the 15 NOD susceptibility
(Idd1-Idd15) alleles (2, 3, 29, 30) (see Materials and
Methods). Unless otherwise stated, only females were used for the
subsequent studies. The CIITA gene is located on chromosome 16 of the
mouse where no Idd susceptibility locus has been mapped (B. Mach,
personal communication). The different genotypes observed were
analyzed for the levels of expression of MHC class II, CD4+
T cells, and CD8+ T cells in the periphery. In agreement
with observations in the nonautoimmune-prone C57/BL6 background (18, 25, 31), MHC class II expression was practically absent in spleen
cells, and there was an almost 10-fold reduction in the levels of
peripheral CD4 T cells in the NOD/CIITA KO mice (Fig. 1B), while in spleen CD8
numbers remained unaltered (Fig. 1C) or slightly
over-represented in the CIITA-deficient mouse (around 25% of total
spleen cells in the CIITA KO vs 16% in the wild-type). This
over-representation of CD8 T cells has also been described in the MHC
class II-deficient mouse (25, 31). This observation confirms that in
the NOD background, CIITA deficiency causes the same defect in positive
selection of CD4 T cells in the thymus as that observed in the C57/BL6
background (18).
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Natural history of the development of diabetes in the NOD/CIITA KO mice
NOD/CIITA-deficient (-/-), heterozygous (+/-), and wt (+/+) mice
were monitored up to 35 wk of age. While in the CIITA-sufficient group
(+/+ or +/-) the incidence of diabetes was approximately 40% by 35 wk
of age, none of the CIITA-deficient (-/-) mice had developed IDDM up
to that age (Fig. 2A) or even
when observed at later times (data not shown). To assess the
possibility that the absence of diabetes in the CIITA KO may be due to
an impairment in the infiltration of the islets, we examined
histological sections taken from NOD/CIITA KO females and prediabetic
or diabetic CIITA-sufficient littermates (Fig. 2B).
Surprisingly, the NOD/CIITA-deficient mice showed pancreatic
infiltration at 15 wk of age. Infiltrates were located predominantly in
the parenchymal tissue and around the vessels (perivascular), and
surrounding the islets (peri-insulitis), seldom infiltrating the islets
(insulitis). Two percent of the islets exhibited some degree of
infiltration, mostly peri-insulitis. In wt NOD mice the infiltrates
were localized mainly around (peri-insulitis) and inside (insulitis)
the islets (75% in total) in both prediabetic and diabetic mice (Fig. 2B). The composition of the infiltrate, as shown by
immunohistochemical analysis of frozen sections, was qualitatively
similar, with the exception of CD4 T cells; CD3-positive T cells, CD8 T
cells, B cells, and macrophages were also present in the pancreatic
infiltrate in CIITA KO mice (Fig. 2C). Thus, the absence of
diabetes in CIITA-deficient mice is not due to an intrinsic defect in
homing and infiltration of the pancreas, although they show much lower
levels of insulitis and a delay in the initiation of infiltration (data
not shown).
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Adoptive transfer experiments were performed to determine whether
the main defect was the near absence of MHC class II-restricted
CD4+ T cells in the periphery and/or the impaired
expression of MHC class II by peripheral APCs. Total spleen cells from
15-wk-old NOD/CIITA-deficient females or prediabetic CIITA wild-type
(wt) littermates were isolated and transferred (15 x
106 cells/recipient) into NOD/SCID recipient females, which
have MHC class II-positive APC, with the exception of B cells. Diabetes
was monitored on a weekly basis up to 56 days posttransfer, and we
observed that while all mice that received splenocytes from prediabetic
wt female mice developed diabetes after the transfer, none of the
recipients of the NOD CIITA KO spleen cells did (Fig. 3
A). This experiment confirmed
the inability of total spleen cells from NOD/CIITA-deficient mice to
cause disease. In contrast, adoptive transfer of prediabetic total NOD
spleen cells into NOD/SCID was able to cause diabetes within
approximately 60 days after the transfer (32). Interestingly, pancreata
from NOD/SCID females, which received NOD/CIITA KO total spleen cells,
did show a small degree of infiltration of the pancreas (data not
shown). This later observation added to the fact that NOD/CIITA KO mice
exhibit pancreatic infiltration strongly suggest that the low
percentage of CD4 cells remaining in the periphery of NOD/CIITA KO mice
is able to promote the incipient infiltration. To address the question
of whether the low levels of CD4 T cells present in the periphery of
NOD/CIITA KO mice are responsible for the pancreatic infiltration
observed in these mice or in NOD/SCID mice, recipients of total spleen
cells from NOD/CIITA KO mice, we performed adoptive transfer
experiments in which NOD/SCID were recipients of total spleen cells or
CD4-depleted spleen cells from NOD/CIITA KO donors (see Fig. 3D, a and b) to monitor CD4 depletion.
As a positive control of the adoptive transfer, NOD/SCID mice were
transferred with total spleen cells from NOD/CIITA-sufficient mice
(positive control recipients). As soon as infiltration was detected in
the positive control recipients (70 days after adoptive transfer), the
other two experimental groups (nondepleted or CD4-depleted
NOD/CIITA KO spleen cells) were bled (to assess the presence of CD4
cells in the periphery) and sacrificed for histological analysis (see
Fig. 3D). Surprisingly we observed that in the CD4-depleted
experimental group the degree of pancreatic infiltration (parenchymal,
perivascular, and perinsular infiltration) was comparable to that in
the nondepleted group (50 and 32%, respectively; see Fig. 3Dd). This result can be potentially explained by the
expansion of some residual CD4 T cells that could not be depleted and
that expand in the presence of nonlymphoid MHC class II+
APCs in NOD/SCID mice. This hypothesis seems to be supported by the
fact that peripheral blood cells isolated from NOD/SCID mice recipients
for nondepleted or CD4-depleted NOD/CIITA KO splenocytes contain a low
percentage of CD4high cells, apparently higher than that
observed in regular NOD/SCID mice (Fig. 3Dc). These results,
although not conclusive, point out a potential role of very low numbers
of CD4 T cells in the initiation of infiltration.
Furthermore, in a complementary approach (Fig. 3B),
when total spleen cells (15 x 106/recipient) from
recently diagnosed diabetic NOD female mice were transferred into 9- to
10-wk-old irradiated NOD/CIITA KO or wt recipients, diabetes was
induced, but the onset of the disease was significantly delayed (9
days; by t test, p < 0.005) in the CIITA KO
recipients compared with that in the wild-type mice. This indicated
that although spleen cells from diabetic mice are already activated,
further local activation in the recipient is required for the
development of disease. Last, but not least, providing NOD/CIITA KO
mice with competent (activated) NOD splenocytes (lymphocytes) rescued
the diabetic phenotype, albeit with retarded kinetics, suggesting that
a deficiency in local activation of the transferred cells causes a
delay in the onset of the disease.
Moreover, further backcrossing of CIITA KO mice onto NOD up to
10 generations (N11) increased the incidence of diabetes of
NOD/CIITA-sufficient mice from 40 to 80%. To exclude the possibility
that further backcrossing would also promote the NOD/CIITA KO mice to
develop diabetes due to unknown Idd susceptibility loci added after
more backcrosses, we performed adoptive transfer experiments in which
total spleen cells from NII NOD/CIITA KO females or NOD/CIITA wt
littermates were transferred into NOD/SCID recipients. As shown in Fig. 3C, total spleen cells from N11 NOD/CIITA KO mice were not
able to transfer disease, while all recipients for NOD/CIITA wt spleen
cells developed diabetes by 70 days after the adoptive transfer. This
result clearly eliminates the possibility that unknown resistant
alleles and not the lack of CIITA expression are responsible for the
absence of diabetes in NOD/CIITA KO mice.
The main lymphocyte defect in the NOD/CIITA KO is in the CD4 T cell subset
To ascertain whether CD8 T cells from the NOD/CIITA KO mice are
functional and, that, when provided with competent CD4 T cell help,
both CD4 and CD8 together can cause diabetes, we performed adoptive
transfer experiments in which NOD/SCID females were used as recipients
for the following: CD4+ T cells from diabetic NOD female
mice, diabetic NOD female CD4+ T cells plus
CD8+ T cells, diabetic NOD female CD4+ T cells
plus NOD/CIITA KO female CD8+ T cells, and
NOD/CIITA-deficient female CD8+ T cells. The results (Fig. 4A) clearly showed that CD8 T
cells from NOD/CIITA KO mice are able to cause disease in the presence
of NOD CD4 T cells, but not in their absence, which again demonstrates
the importance of CD4 T cells in the pathogenesis of diabetes. The near
absence of peripheral MHC class II-restricted CD4 T cells in the
NOD/CIITA KO mice may lead to an impairment in the activation of the
different effector populations, like that in CD8 T cells.
Interestingly, when pancreata from NOD/SCID recipients of NOD/CIITA KO
CD8 T cells were examined, no infiltration was observed (Fig. 4B), indicating that CD4 T cells are also necessary for the
initiation of infiltration in adoptive transfer. This hypothesis is
supported by the fact that the recipients of NOD CD4 T cells alone
showed a high degree of infiltration (see Fig. 4B), almost
comparable to that observed in the recipients of both CD4 and CD8 T
cells, although no diabetes was observed when transferring CD4 T cells
alone. The slight delay observed in the onset of diabetes when a
mixture of NOD CD4 T cells and NOD/CIITAKO CD8 T cells was transferred
is statistically significant (by t test, p
< 0.001). This delay may be due to the fact that CD8 cells obtained
from diabetic NOD females as donors are already activated, while those
from NOD/CIITA KO mice have to be activated upon cotransferance with
the NOD CD4 T cells into the host.
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To confirm that NOD/CIITA KO CD8 T cells are functional and to
assess whether they show a Tc2-like phenotype that might protect these
mice from diabetes we performed in vitro activation studies on
FACS-sorted CD3+CD8+ T cells from spleen either
from NOD/CIITA-sufficient (+/-; 18 wk old, on the average) or KO (19
wk old, on the average) mice. Cells were stimulated in vitro with Con A
on day 0 and at 20 h (day 1) were tested for surface expression of
CD69 and CD62L (Fig. 5A) as
markers to assess the level of activation. On day 3 cells were pulsed
with [3H]thymidine and harvested on day 4 (data not
shown). In parallel, a separate batch of cells was restimulated with
Con A, and supernatants were collected on day 5 to analyze cytokine
production (IFN- and IL-4). The degree of up-regulation of CD69
(expressed as the increase over the nonstimulated control value) upon
activation with Con A was similar in both populations (wt and KO) of
CD8 T cells (between 1.6-fold in the KO and 2.3-fold in the wt; Fig. 5A). Similarly, CD62L expression was down-regulated to the
same extent in CD8 T cells from both wt and KO mice (1.5- and 1.3-fold,
respectively; Fig. 5A). Both populations proliferated
equally in response to Con A stimulation (data not shown), and
interestingly, the cytokine profile was the same: high production of
IFN- (almost 400 U/ml; Fig. 5B) was seen, while no IL-4
production was detected (data not shown). This observation suggested
that in vivo CD8 T cells in the NOD/CIITA KO probably have a Tc1
phenotype comparable to that seen in the CIITA-sufficient mice. There
does not, therefore, appear to be an intrinsic defect in CD8 T cells
that prevents them from being activated in NOD/CIITA-deficient mice.
The results shown in Fig. 4A clearly indicate that CD8 T
cells in NOD/CIITA KO mice need CD4 T cell help to be activated and to
be able to promote diabetes.
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Another possible deficiency in NOD/CIITA KO mice could be that B
cell function, which is also involved in the pathogenesis of diabetes
(30), could be impaired due to the lack of MHC class II expression. We
isolated B220+ cells by FACS and stimulated them in vitro
with LPS in the presence or the absence of IL-4 on day 0. On day 1,
some of the cells were harvested to check the expression of activation
markers on the surface (CD23, B7.1, B7.2, MHC class II). Up-regulation
of CD23 and B7.2 was observed in B cells from both CIITA-sufficient and
-deficient mice only when IL-4 (20 U/ml) was also present in the
culture medium (Fig. 5C). No up-regulation of CD40 (data not
shown) and B7.1 was observed in either B cells of CIITA-sufficient or
-deficient mice (Fig. 5C), and only in the
NOD/CIITA-sufficient mice was up-regulation of MHC class II observed
upon stimulation (Fig. 5C). The level of proliferation upon
LPS stimulation was the same in both B cell populations (data not
shown). We also measured Ig production in the presence or the absence
of LPS, with or without IL-4 in the culture medium. Supernatants were
collected after 7 days and were assayed by ELISA for the presence of
IgM, IgG1, IgG2a, IgG2b, and IgG3. The values obtained for all the Igs
tested are similar in the CIITA-sufficient and -deficient mice (data
not shown). There is, therefore, no intrinsic defect in B cell function
in the NOD CIITA-deficient mice.
The autoaggressive CD8+ T cell clone TGNFC8 is able to cause IDDM when transferred into NOD/CIITA KO recipients
The TGNFC8 CD8 T clone exhibits an autoaggressive phenotype, since
it can cause disease when transferred in the absence of CD4 T cells
into NOD/SCID or irradiated NOD recipients (13, 33). We questioned
whether this would be the case in NOD/CIITA KO mice, which have a
deficiency in CD4 T cell help. CD8 T cells were adoptively transferred
into nonirradiated 3- to 4-wk-old NOD recipients (107
cells/mouse). The CD8+ T cell clone evoked diabetes in
NOD/CIITA KO recipients, although with a significant delay (by
t test, p < 0.05) compared with that in
NOD/CIITA-sufficient (+/-) recipients (Fig. 6A). This outcome highlights
the fact that high numbers of activated autoaggressive CD8 T cells can
over-ride the need for CD4 T cell help. The delay in the onset of
diabetes in NOD/CIITA KO recipients may be explained by the requirement
of CIITA for IFN--induced MHC class I expression (22, 23). This
delay is also observed when irradiated N11 NOD/CIITA KO mice were
recipients of the TGNFC8 CD8 cloned T cells (Fig. 6B) and
compared with irradiated N11 NOD/CIITA-sufficient recipients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Moreover, adoptive transfer experiments using CD4 T cells alone
isolated from diabetic NOD females show that activated CD4 T cells
alone (Fig. 4B) are able to infiltrate the pancreas. On the
other hand, ß2m KO mice on the NOD background exhibit no
infiltration (11, 14), which clearly suggests that CD8 T cells are
required at the beginning for the priming of CD4 T cells.
Interestingly, recent work shows that there are residual class II
+ cells in the thymus and in s.c. lymph nodes in
CIITA-deficient mice (dendritic cells in inguinal s.c. lymph nodes
express MHC class II, although at fivefold reduced levels compared with
those from mice heterozygous for the mutation) (24). These class II
+ cells may account for the 5% CD4-positive cells observed
in the periphery (by providing some degree of positive selection in the
thymus) and the pancreatic infiltration observed in the NOD/CIITA KO
mice (Ag presentation by class II+ APCs in the periphery).
However, those low levels of MHC class II expression are not enough to
promote insulitis and diabetes. This is also supported by the
significant delay observed in the development of disease following
adoptive transfer of total spleen cells from diabetic NOD mice into
irradiated NOD/CIITA KO mice compared with irradiated
NOD/CIITA-sufficient mice (Fig. 3B). The deficiency in MHC
class II itself may be detrimental for the local activation of the
different lymphocytic populations. IFN- induces MHC class II
expression in a variety of different cell types, for instance cells of
the macrophage-monocyte lineage, endothelial cells, epithelial cells,
fibroblasts, and muscle cells (19, 36, 37). As a consequence,
endothelial cells can become competent APCs, as has been reported for
human vascular endothelium, being more efficient in presenting Ags to
resting memory T cells than to naive T cells (37). Thus, the lack of
CIITA may prevent IFN--induced MHC class II expression in pancreatic
endothelial cells, causing a delay in the transfer of disease by the
activated CD4 +T cells existing in the spleen from the
diabetic donor.
While the paucity of CD4 T cells in the CIITA KO mice probably
explains the phenotype, these results do not exclude a role for the
residual CD4 T cells that are found in both these mice and class II KO
mice. In fact, our results are consistent with a role for the remaining
CD4 T cells in promoting pancreatic infiltration. Specifically, when
CD8 T cells from NOD/CIITA KO mice are transferred into NOD/SCID mice
no infiltration is observed, while CD4 T cells from diabetic NOD
females infiltrate the pancreata from NOD/SCID recipients without
causing disease. Moreover, we have also observed that total spleen
cells from NOD/CIITA KO mice promote infiltration, with no diabetes
induction in NOD/SCID recipients (data are shown for the N11 generation
in Fig. 3Dc). On the other hand, the results obtained in
adoptive transfer experiments of CD4-depleted spleen cells from
NOD/CIITA-deficient donors into NOD/SCID mice are not conclusive, since
pancreatic infiltration remained, but a compensatory increase in CD4 T
cells occurred posttransfer. These studies may indicate 1) an important
potential role for low numbers of CD4 T cells in the initiation of
pancreatic infiltration; and 2) that caution should be taken in the
interpretation of CD4 depletion experiments, since there is no absolute
guarantee of complete blocking or removal (depletion) of CD4 cells, and
very few residual cells may be able to undergo compensatory
proliferation in the presence of MHC class II+ cells.
The infiltration observed in the NOD/CIITA-deficient mouse is
consistent with the major postulated role of the CD8 T cell subset in
these mice in promoting insulitis (12, 28). Reconciling these
observations, we propose that CD4 T cells are involved in the
initiation of the infiltration together with CD8 T cells. This is
consistent with the less severe degree of infiltration (mostly
perivascular and parenchymal) observed in NOD/CIITA KO mice compared
with that in wild-type littermates. This may be seen in terms of CD4 T
cells facilitating the initial entry of CD8 T cells into the islet to
cause early damage to the ß-cells. CD4 T cells are also necessary
later in the final effector phases. Furthermore, in the absence of MHC
class II expression, B cells cannot be activated by CD4 T cells, whose
numbers, in turn, are very low in NOD/CIITA-deficient mice. The
constitutive expression of MHC class II in B cells is dependent on
CIITA, whose expression in these cells is driven by the CIITA promoter
called PIII (18, 20, 38). However, the up-regulation of MHC class II
depends not on B cells on IFN- but, rather, on other cytokines, such
as IL-4, IL-10, or Abs against surface Ags (i.e., 
IgM, IgD,
Lyb2, or B220) (39, 40, 41, 42, 43, 44). IFN- has a down-regulating effect on
MHC class II expression by B cells (45). B cells may play a role in the
precipitation of disease, since total spleen cells from NOD mice
transferred into NOD/SCID cause disease more rapidly that isolated T
cell populations (32).
CD4 T cell help can be over-ridden when high numbers (10 x
106 cells/mouse) of in vitro activated cloned
autoaggressive CD8 T cells are transferred into NOD/CIITA KO
recipients. This fact emphasizes that numbers of cells are of
importance in the pathogenesis of diabetes. However, the delay in the
kinetics of onset of the disease suggest that there is also an
impairment in the local activation of autoaggressive transferred CD8 T
cells in NOD/CIITA KO mice. This delay, also observed in further
backcrossed NOD/CIITA KO mice (N11), may be due to impaired IFN-
induction of MHC class I in the NOD/CIITA KO recipients’ pancreata.
This seems, however, to be in conflict with a recent report showing
that peritoneal macrophages obtained from CIITA KO mice do not exhibit
impaired MHC class I up-regulation upon IFN- treatment. Since all
nucleated cells express MHC class I, however, it is possible that
regulation of MHC class I expression by these cell types may be
different and in some cases CIITA dependent.
The cumulative incidence of diabetes in CIITA-sufficient littermates
(+/+ or +/-) was 40% (at the fifth backcross with 15 NOD alleles
fixed), and a slight delay in the time of onset of the disease was
observed. In our NOD colony the incidence in females is about 90% by
30 wk of age. After nine backcrosses onto the NOD background, the
cumulative incidence observed in NOD/CIITA-sufficient mice was 80% by
30 wk of age, with no significant delay compared with our NOD colony
(data not shown). After further backcrossing, total spleen cells from
N11 NOD/CIITA KO mice are still unable to transfer diabetes into
NOD/SCID recipients. This excludes the possibility that unknown
resistant Idd loci, and not the CIITA gene, are the responsible for the
absence of diabetes in N6 NOD/CIITA KO mice (Fig. 3C).
It has been postulated than the functional deficiency of NK1-like T
cells in the NOD mouse leads to an impairment in Th2 cell function,
leading to a Th1 phenotype (46, 47). According to this model these NK T
cells would be the main producers of IL-4 upon initial stimulation
(48). One could hypothesize that in NOD/CIITA-deficient mice this
NK T cell population would be enriched in the periphery due to the
absence of MHC class II-restricted CD4 T cells and that this would
protect from diabetes. The remaining CD4+ cells in the
periphery of MHC class II-deficient mice (5%) are, however, also
present in ß2m KO MHC/class II KO double-deficient mice
(31). This indicates that those residual cells are not CD1 restricted
NK T cells, which are poorly selected in the absence of
ß2m. In NOD/CIITA-deficient mice we have observed that in
the spleen the remaining CD4+ cells are mostly
CD3+. The low number of this T cell subset makes their
study difficult. CD8 T cells obtained from NOD/CIITA-deficient mice
appear to be normal and do not inhibit the activated NOD CD4 T cells
when cotransferred onto NOD/SCID to cause diabetes. This argues against
a protective Tc2 phenotype conferred by increased IL-4 production. In
addition, CD8 T cells, when activated in vitro, show a clear Tc1
cytokine production pattern: large amounts of IFN- are produced,
while IL-4 is not detectable. Moreover, we have performed experiments
in which total spleen cells from NOD/CIITA KO mice have been
adoptively transferred into 3-wk-old NOD females and compared with
controls in which total spleen cells from NOD mice were transferred
into NOD recipients. We did not observe any delay in the onset of
diabetes in recipients of NOD/CIITA KO total spleen cells compared with
that in recipients of NOD total spleen cells. This observation clearly
suggests that there is no protective (IL-4-related?) phenotype for
those cells coming from NOD/CIITA KO when transferred into NOD
recipients (data not shown).
In addition, the interaction between CD40L on T cells and CD40 on APCs has been shown to play a crucial role in the activation of both APCs and T cells (49). CD40 is expressed in B cells, monocytes, dendritic cells, hemopoietic progenitors, and epithelial cells; CD40L is expressed in activated T cells, mostly CD4 T cells, but also some CD8 T cells (50, 51). For APCs CD40 ligation constitutes a survival signal (52) and up-regulates the expression of costimulatory molecules such as B7.1 and B7.2, adhesion molecules (ICAM-1), and MHC class II (53, 54). Upon CD40 ligation endothelial cells up-regulate ICAM-1, E-selectin, and VCAM-1 (55). On the other hand, CD40L-CD40 interactions enhance T cell priming (56), and are necessary to provide T cell help to B cells (57, 58, 59). It is generally assumed that the main source of CD40L is activated CD4 T cells, although CD8 T cells may be also primed indirectly through CD40 signaling to the APC (60). Then in NOD/CIITA KO mice, the near absence of CD4 T cells, and therefore the main source of CD40L, may lead to a scenario in which activation of APCs (absence of MHC class II and CD40 ligation) and endothelial cells (failure of up-regulation of MHC class II and adhesion molecules) and indirect priming of CD8 T cells (60, 61, 62) (through either activated APCs and/or CD4 T cells) are severely impaired. That these effects are likely to be important contributions of CD4 T cells is supported by the recent observation that anti-CD40L Abs block diabetes and insulitis (63).
From the present study we conclude that the main defect in the development of diabetes in NOD/CIITA-deficient mice is the practical absence of functional MHC class II-restricted CD4 T cells, which would provide help for the initial infiltration into the islets (insulitis) and for the different effector mechanisms involved in ß-cell destruction. The precise nature of these mechanisms remains a topic for further study.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard A. Flavell, Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address:
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; CIITA, class II transactivator; KO, knockout; wt, wild type; CD40L, CD40 ligand.
Received for publication August 26, 1998. Accepted for publication January 27, 1999.
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