From the Department of Immunology (M.E.P., A.N., D.L.) and the Department of Neuropharmacology (T.W., M.v.H.), Division of Virology, The Scripps Research Institute; Integrative Biology (D.L.), Digital Gene Technologies, La Jolla, California; the Department of Immunology and Infectious Diseases (I.-C.H., L.H.G.), Harvard School of Public Health; and the Department of Medicine (L.H.G.), Harvard Medical School, Boston, Massachusetts. The present affiliation for M.E.P. is the Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois.
Address correspondence and reprint requests to Dr. David Lo, Integrative Biology, Digital Gene Technologies, 11149 North Torrey Pines Rd., Suite 110, La Jolla, CA, 92037. E-mail: davidlo{at}dgt.com .
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ABSTRACT |
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INTRODUCTION |
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Whereas these disease models may differ in kinetics, T-cell subset involvement, and inducing factors, other aspects remain similar. These include the activation of autoreactive T-cells and the development of highly structured islet infiltrates before clinical hyperglycemia (8,11,12,13,14). These characteristics are also shared with human diabetes, making them useful models for uncovering disease mechanisms and helpful for the evaluation of potential treatments, such as cytokine and gene therapies (15,16).
Acceleration of autoimmune diabetes has been associated with T helper 1 (Th1) cells (17,18,19,20), whereas resistance has been associated with increased T helper 2 (Th2) cytokines (21,22,23,24,25,26,27). Thus, one immunotherapeutic approach for diabetes is to introduce genetic manipulations that skew T-cell responses toward Th2 cytokine production. For example, recent work suggests that transgenic interleukin (IL)-10 overproduction by islet-specific T-cells can provide limited protection from diabetes (28). Alternatively, genetic manipulation of key transcription factors may provide a more efficient way of controlling cytokine production. Because IL-4 is pivotal for Th2 cell development (29,30) and its increase is also associated with diabetes inhibition (31,32), transcription factors that control IL-4 transactivation, such as c-Maf (33,34,35), are of particular interest. Fortunately, c-Maf transgenic T-cells do not constitutively produce IL-4, but instead overexpress IL-4 rapidly after T-cell receptor-mediated stimulation (34). So in effect, antigen-inducible expression of IL-4 may be accomplished through constitutive production of the transcription factor c-Maf.
Here we test the ability of transgenic c-Maf to attenuate autoimmune diabetes using three different models of disease. In two of these models (TCR-SFE/Ins-HA and RIP-LCMV-NP), constitutive T-cell expression of c-Maf leads to significant inhibition of disease. This corresponds to c-Mafmediated early increases in type 2 cytokine production by activated T-cells. Interestingly, diabetes was not attenuated in c-Maf transgenic NOD mice, suggesting that additional complex genetic factors influence the ability of c-Maf to affect disease.
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RESEARCH DESIGN AND METHODS |
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Mouse genotyping. Standard PCR analysis of tail DNA was used to determine mouse genotypes. To identify homozygous H-2d (B10.D2) mice, the micro-satellite marker MM23 primers (sense: 5'-GTTTCAGTTCTCAGGGTCCTA-3' and anti-sense: 5'-CAGGATTCTGTGGCAATCTGG-3') were used. The following primers were used to identify integration of the indicated transgenes: c-Maf, sense: 5'-TGTTGTGGTGCAGAACTGGAT-3' and anti-sense: 5'-GTTTCA GGTTCAGGGGGAGGT-3'; TCR-SFE, sense: 5'-GAACTGCTCAGCATAACT CCC-3' and anti-sense: 5'-GAGGCTGCAGTCACCCAAAG-3'; Ins-HA, sense: 5'-CAATTGGGGAAATGTAACATCGCCG-3' and anti-sense: 5'-AGCTTTGGGTAT GAGCCCTCCTTC-3'. Genotyping of RIP-LCMV-NP mice was performed by hybridization of tail DNA with NP-specific probes as previously described (7).
Adoptive transfer and viral induction models. For adoptive transfer models of disease, spleen and lymph node mononuclear cells were pooled from c-Maf/TCR-SFE or TCR-SFE mice. The total number of CD4+ T-cells in each pool was determined by multiplying the total cell yield with the percent CD4+ cells as determined by flow cytometry using anti-CD4-phycoerythrin (PharMingen). Adoptive transfers of 1 x 107 CD4+ T-cells were performed by intravenous injections into irradiated (700 rad) Ins-HA recipients. The total cell numbers varied for each donor group, but CD4 T-cell numbers were kept constant for each recipient injection. Blood glucose levels were monitored weekly after cell transfer. In a second adoptive transfer model, 5 x 106 purified CD4+ cells from spleen and lymph nodes of age-matched diabetic TCR-SFE/Ins-HA or nondiabetic c-Maf/TCR-SFE/Ins-HA mice were injected intravenously into nonirradiated RAG-1-/-/Ins-HA recipients. Blood glucose levels were monitored every 3-4 days post-transfer. Diabetes induction of 6- to 8-week-old c-Maf+or-/RIP-LCMV-NP mice was accomplished by intraperitoneal injection of 105 PFU of LCMV Armstrong, clone 53b (37). Blood glucose levels were monitored weekly after infection.
Blood glucose monitoring and statistics. Onset of diabetes (>16.6 mmol/l) was determined by monitoring blood glucose levels using Chemstrip bG test strips with an Accu-Chek III blood glucose monitor (Boehringer Mannheim). Incidence of diabetes was graphed on Kaplan-Meier cumulative survival plots, and the log-rank (Mantel-Cox) test for statistical significance was performed using StatView software.
Histology. Pancreas tissue was harvested, fixed in zinc formalin, and paraffin embedded before sectioning. Sections were stained with periodic acid-Schiff (PAS) by standard methodology.
T-cell purification. CD4+ or CD8+ T-cells were purified from lymph nodes using negative selection with magnetic beads. Briefly, lymph nodes were dissociated into single-cell suspensions, passed through 70-µm cell strainers, and washed with RPMI-1640 plus 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l glutamine, 25mmol/l HEPES, and 5 x 10-5 mmol/l ß-mercaptoethanol (complete medium). Cells were incubated for 30 min at 4°C in phosphate-buffered saline containing 2% serum and 2 µg/ml rat antimouse B220 (PharMingen) plus either 2 µg/ml rat antimouse CD8 (PharMingen) for CD4 cell purification or 2 µg/ml rat antimouse CD4 (L3T4, PharMingen) and 2 µg/ml rat antimouse CD4 (YTS.177) for CD8 cell purification. Then cells were washed and undesired cell populations were removed using BioMag goat antirat IgG (H+L)conjugated magnetic beads (Polysciences) according to the manufacturer's instructions.
Proliferation assays and enzyme-linked immunosorbent assay. Lymph
node mononuclear cells were isolated from 6- to 8-week-old c-Maf, TCR-SFE,
NOD/c-Maf, or NOD mice. For proliferation assays, 3 x 105
lymph node CD4 or CD8 cells/well (purified by negative selection) were
stimulated with plate bound antimouse CD3 (10 µg/ml;
PharMingen) plus anti-mouse CD28 (1 µg/ml; PharMingen) for 24 h and pulsed
with 1 µCi/well for another 24 h before harvest. Triplicate samples were
counted on a Microbeta Trilux liquid scintillation counter (Wallac).
For cytokine analysis, 3 x 105 CD4+ or
CD8+ lymph node cells from 6- to 8-week-old mice were stimulated as
described above in 200 µl/well complete medium. Supernatants were collected
after 48 or 72 h and assayed for IL-4, IL-5, IL-10, or -interferon
(IFN-
) by sandwich enzyme-linked immunosorbent assay (ELISA) using the
appropriate antibody pairs (PharMingen) and peroxidase-conjugated streptavidin
plus 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). All
incubations were for 30 min at 37°C. Plates were washed with
phosphate-buffered saline containing 0.05% Tween 20 after each incubation.
Cytokine concentrations were interpolated from standard curves obtained from
spectrophotometer (Spectra 2.06) readings using DeltaSoft 3 software. For
analysis of secondary responses, cells were stimulated 72 h then rested in
complete medium with 20 U/ml rhIL-2 (Pepro Tech, Rocky Hill, NJ) for 4-5 days.
Cells were then restimulated at 3 x 105 cells/well (200
µl/well) and supernatants were harvested after 24 h and assayed for
cytokine levels as described above.
Cytotoxic T-lymphocyte (CTL) assays and pCTL frequency. For analysis of primary CTL activity, c-Maf transgenic or nontransgenic littermate mice were injected intraperitoneally with 105 PFU of LCMV. Effector cells were obtained from spleens harvested 7 days postinfection. CTL activity was assessed by standard 51Cr release assays using uninfected or LCMV-infected H-2d fibroblasts (BALB C17) as target cells at 100:1 and 50:1 effector/target ratios. Triplicate cultures were incubated for 5 h at 37°C with 5% CO2 as previously described (7). For analyses of CTL activity after secondary stimulation, splenocytes were harvested 30-60 days postinfection (as described above) and cultured in vitro for 1 week with syngeneic LCMV-infected macrophages and 50 U/ml IL-2. Here primary stimulation occurred in vivo, whereas the secondary stimulation was performed in vitro. CTL activity was assessed as described above using effector:target ratios of 10:1 and 5:1. Precursor CTL frequencies were determined as previously described (37). Briefly, splenocytes were harvested 7 days after LCMV immunization and serially diluted in 96-well flat bottom plates containing LCMV-infected irradiated (2,000 rad) macrophages and irradiated syngeneic feeder splenocytes. After 8 days, each well was tested with 51Cr-labeled LCMV-infected BALB C17 target cells in a standard 5 h 51Cr release assay. CTL precursor frequencies were calculated as follows: pCTL(f) = (4.6-ln[percentage of negative wells])/number of splenocytes per well). The pCTL frequencies were defined as the slope of the linear regression along at least three separate data points. Positive cultures were defined by a specific 51Cr release more than three standard errors above background lysis.
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RESULTS |
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The influence of c-Maf on diabetes onset was initially evaluated using the TCR-SFE/Ins-HA transgenic model of spontaneous diabetes in which disease is mediated by antigen-specific CD4 cells (8). Here CD4 T-cells bearing the major histocompatibility complex (MHC) class II restricted T-cell receptor TCR-SFE are specific for a hemagglutinin peptide that is expressed by islet ß-cells (Ins-HA transgenic mice). TCR-SFE/Ins-HA mice develop aggressive disease, and 100% are diabetic within 6 weeks of life (Fig. 2A). Strikingly, the onset of disease is significantly delayed and the overall incidence decreased in mice that carry the c-Maf transgene (Fig. 2A). Thus, constitutive T-cell expression of c-Maf leads to inhibition of diabetes in this CD4-dependent model of spontaneous disease.
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c-Maf does not inhibit T-cell proliferation, insulitis, or adoptive transfer of disease. As with CD4 cells from c-Maf single transgenic mice, type 2 cytokine production is increased among CD4 cells from c-Maf/TCR-SFE double transgenic mice indicating that the TCR-SFE transgene does not adversely influence the effect of c-Maf (Fig. 2B). Reduced disease incidence among c-Maf/TCR-SFE/Ins-HA mice is probably not the result of direct c-Mafmediated inhibition of T-cell proliferation because CD4 T-cells from c-Maf transgenic mice do not differ from nontransgenic littermates in proliferative responses to anti-CD3 plus anti-CD28 (Fig. 2C). Furthermore, the accumulation of lymphocytes in the pancreatic islets of Langerhans appears unchanged by c-Maf as judged by the incidence and severity of peri-insulitis (Fig. 3).
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Diabetes also occurs after passive transfer of TCR-SFE transgenic CD4 T-cells into Ins-HA mice (8,12,28). Disease onset in this transfer model is rapid (within 5 weeks of transfer) but requires lymphocyte-depleted recipients, so the mechanisms leading to disease here may differ from the spontaneous disease seen in TCR-SFE/Ins-HA transgenic mice (12,28). In contrast to the protection afforded by c-Maf in the spontaneous transgenic model, c-Maf does not have a significant effect on disease onset mediated by adoptively transferred SFE-specific CD4 cells (Fig. 2D). Furthermore, CD4 cells from nondiabetic c-Maf/TCR-SFE/Ins-HA mice also cause disease when transferred into RAG-1-/-/Ins-HA recipients (Fig. 2E). While c-Maf changes the kinetics of type 2 cytokine expression and inhibits disease in the TCR-SFE/Ins-HA transgenic model of spontaneous diabetes, it cannot prevent disease mediated by adoptively transferred T-cells.
c-Maf also inhibits virus-induced diabetes in RIP-LCMV-NP mice. To test the effect of c-Maf in an inducible model of diabetes, we used the well-characterized RIP-LCMV-NP disease model (6,7,27,37,38,39). RIP-LCMV-NP mice express the nucleoprotein of LCMV in the thymus and islet ß-cells and only become diabetic after infection with LCMV (typically within 8 weeks postinfection). c-Maf transgenic RIP-LCMV-NP mice are significantly more resistant to disease than the control littermate mice (Fig. 4A).
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To investigate whether disease attenuation might be attributable primarily to the c-Mafmediated change in CD4 helper function (Fig. 1) or might involve additional changes in CD8 effector functions, we examined the CTL activity of c-Maf transgenic T-cells. c-Maf transgenic or nontransgenic littermates were primed in vivo with LCMV (105 PFU) 7 days before the harvest of spleens for analysis of primary antigen-specific CTL activity. For analysis of memory CTL activity, spleens were harvested 30-60 days postinfection and then restimulated in vitro before analysis. Both the primary and memory virus-specific CTL responses of c-Maf transgenic mice were reduced compared with nontransgenic littermates (Fig. 4B). We also examined lytic precursor frequencies after in vivo infection with LCMV in c-Maf+or-/RIP-LCMV-NP mice. LCMV-specific precursor CTL frequencies were significantly reduced in c-Maf/RIP-LCMV-NP mice compared with non-transgenic littermate controls, 1/18,000 (± 5,000) and 1/3,200 (± 1,500), respectively. Although disease attenuation in the RIP-LCMV-NP model parallels the c-Maf-mediated increase in type 2 cytokine production (Fig. 1), it also reflects a reduction in CTL development and a reduced precursor frequency.
Despite its ability to increase IL-4 cytokine production, c-Maf does not delay disease onset in NOD mice. Spontaneous autoimmune diabetes is typically a multigenic disorder (40,41,42). Both TCR-SFE/Ins-HA and RIP-LCMV-NP models are on the B10.D2 background. Studies have shown that this genetic background, closely related to C57BL/6 and C57BL/10, is permissive for autoimmune disease in transgenic models (13,42). In comparison, the NOD mouse strain is permissive for spontaneous autoimmune diabetes even in the absence of transgenes (41,42). Because diabetes in NOD mice is also thought to be driven by Th1 effectors (18), we tested whether the c-Maf transgene could protect NOD mice from diabetes. After backcrossing the c-Maf transgene five generations to NOD, we found that nontransgenic littermates developed diabetes at a high frequency (Fig. 5). Surprisingly, the presence of the c-Maf transgene had no protective effect; indeed, there appears to be a slight acceleration of disease that is not quite statistically significant (P = 0.0515; Fig. 5). Although the c-Maf transgene caused some increased IL-4 expression in CD4 T-cells after stimulation with anti-CD3 plus anti-CD28 (Fig. 6), the effect was not as strong as that seen in CD4 cells from B10.D2 mice (compare Figs. 1 and 6). Type 2 cytokine upregulation was also not significantly increased among NOD/c-Maf transgenic CD8 cells.
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DISCUSSION |
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In the TCR-SFE/Ins-HA transgenic model of spontaneous diabetes, c-Maf significantly inhibits disease. Because diabetes in this model is dependent on MHC class II restricted CD4 T-cell effector function (8), disease inhibition is most likely a consequence of the c-Maf-mediated early shift in type 2 cytokines produced by antigen-specific CD4 cells. c-Maf had no significant direct effects on T-cell proliferation in vitro or peri-insulitis in vivo, although current studies cannot rule out unusual skewing of T-cell populations within the islet tissue itself. Thus, regulated overexpression of multiple type 2 cytokines by antigen-specific T-cells can significantly inhibit diabetes onset. Although we have previously demonstrated that the c-Maf transgene induces global changes in cytokine expression by T-cells, the effect on diabetes in the TCR-SFE/Ins-HA model is likely caused by the local effects within islet infiltrates as TCR-SFE T-cells are triggered to express type 2 cytokines by presentation of HA in and around islets. Notably, disease attenuation is much more effective with transgenic c-Maf than previously observed with regulated T-cell overexpression of IL-10 alone (28).
Although a number of studies show that type 2 cytokines can inhibit
autoimmune diabetes
(21,22,23,24,25,26,27,28),
it is clear that profound immune deviation does not always alleviate disease.
NOD mice with a targeted disruption in the IFN gene are
unable to generate classical Th1 cells, yet still become diabetic
(43). This strong skewing is
avoided in c-Maf transgenic mice in which constitutive T-cell expression of
c-Maf leads to increased production of multiple type 2 cytokines but not
complete elimination of Th1 cells
(34). While c-Maf-mediated
upregulation of type 2 cytokines is effective in attenuating disease in our
TCR-SFE/Ins-HA model of spontaneous disease, it cannot inhibit disease onset
after the adoptive transfer of islet-specific CD4 cells, even from previously
protected mice. This suggests that the modest increases in type 2 cytokines
afforded by c-Maf may be insufficient to block disease when it is mediated by
antigen-specific CD4 cells once they have been activated.
Virus-induced diabetes is also significantly attenuated by transgenic
c-Maf. Because diabetes after LCMV infection of RIP-LCMV-NP requires both
antigen-specific CD4 and CD8 participation
(7,38),
disease mechanisms are more complex here than in TCR-SFE/Ins-HA mice. The
comparison of cytokine responses in CD4 and CD8 cells demonstrates that c-Maf
can direct increases in type 2 cytokines in both cell types. Having
established that c-Mafmediated changes in CD4 cells lead to significant
attenuation of diabetes in TCR-SFE/Ins-HA mice, it became important to
determine if c-Mafmediated disease attenuation is primarily the result
of changes in CD4 effector function or if additional CD8 effector functions
are altered. Analysis of antigen-specific CTL activity shows that c-Maf
mediates an 2-fold reduction in antigen-specific killing and a 5- to
6-fold decrease in CTL precursor frequency. Previously we have shown that IL-4
can suppress CTL activity indirectly through a STAT-6dependent effect
on antigen-presenting cells
(27). Given the increased IL-4
production capacity of c-Maf transgenic T-cells, a similar mechanism may lead
to the reduced CTL activity observed in c-Maf/RIP-LCMV-NP mice. It is also
possible that other more direct c-Mafmediated changes in T-cell
function may occur that impact disease onset. One possibility is a change in
migration ability, particularly because unique in vivo migration kinetics have
been observed for Tc1 versus Tc2 cells that correspond to altered viral
clearance abilities (44).
However, viral clearance was not affected by transgenic c-Maf as determined by
viral plaque assays (37) (data
not shown). The current data indicate that changes in both CD4 and CD8
effector functions could account for attenuation of diabetes after viral
infection of c-Maf/RIP-LCMV-NP mice.
While the effectiveness of transgenic c-Maf is impressive in the TCR-SFE/Ins-HA and RIP-LCMV-NP models in which disease incidence can be reduced by >60%, it has no inhibitory effect among NOD mice. It appears that the Th1-favoring genetic predisposition of NOD mice is not easily skewed by c-Maf. In fact, a trend toward accelerated disease onset occurs in the presence of c-Maf, but this is not statistically significant. This effect is not likely the result of an overwhelming allergic inflammation (21); islet infiltrates in c-Maf/NOD mice are histologically similar to NOD littermate controls (data not shown), lacking the abscess formation and profound eosinophilia that has been observed in some cases after transfer of diabetogenic Th2 cells (21). Alternatively, c-Maf could potentially influence antigen-presenting cell function indirectly through cytokine regulation, as has been suggested for IL-4 (27); however, this awaits further investigation. How such changes would affect disease onset among different mouse strains and disease models is currently unclear.
The differing results obtained from the combined evaluation of transgenic c-Maf using multiple disease models illustrates the need for caution when evaluating therapies for use among genetically diverse clinical populations. In addition, results here also suggest logical focal points for uncovering functional immune defects that might contribute to the complex multigenic susceptibility of diabetes in NOD mice. For example, the resistance of NOD mice to changes in T-cell function mediated by transgenic c-Maf suggests that the coordinate action of factors that directly regulate the IL-4 gene may differ among susceptible and resistant mouse strains. Alternatively, differences in other factors involved in IL-4 receptor signaling might promote the Th1 predisposition of NOD mice and in this way also contribute to disease susceptibility.
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ACKNOWLEDGMENTS |
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We thank Christina Reilly, Lian Fan, and Heather Neal for their excellent technical assistance.
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FOOTNOTES |
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CTL, cytotoxic T-lymphocyte; ELISA, enzyme-linked immunosorbent assay; HA,
hemagglutinin; IFN-,
-interferon; IL, interleukin; LCMV,
lymphocytic choriomeningitis virus; MHC, major histocompatibility complex; NP,
nucleoprotein; PAS, periodic acid-Schiff; PCR, polymerase chain reaction; RIP,
rat insulin promoter; TCR, T-cell receptor; Th1, T helper 1; Th2, T helper 2;
TSRI, the Scripps Research Institute.
Received for publication March 22, 2000 and accepted in revised form September 26, 2000
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REFERENCES |
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