Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0406252 on December 12, 2006

Published online before print December 12, 2006
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(Journal of Leukocyte Biology. 2007;81:757-765.)
© 2007 by Society for Leukocyte Biology

Distinct regulation of autoreactive CD4 T cell expansion by interleukin-4 under conditions of lymphopenia

Natasha J. Hill, Aleksandr B. Stotland and Nora E. Sarvetnick1

The Scripps Research Institute, La Jolla, California, USA

1 Correspondence: The Scripps Research Institute, 10550 N. Torrey Pines Road, IMM-23, La Jolla, CA 92037, USA. E-mail: noras{at}scripps.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-4 is protective against Type 1 diabetes in the NOD mouse. IL-4 promotes T cell survival in vitro, but little is known about the effect of IL-4 on clonal expansion in vivo. Here, we show that IL-4 only enhances the expansion of autoreactive CD4 T cells during lymphopenia and that neither the presence of islet IL-4 nor IL-4 deficiency affects T cell expansion significantly under conditions of immunosufficiency. The accumulation of proliferating cells induced by IL-4 in a lymphopenic host is inhibited incrementally by increasing the number of bystander cells and is prevented by cell numbers well below that of unmanipulated NOD mice. The ability of IL-4 to promote autoreactive CD4 T cell expansion is therefore sensitive to the degree of host immunodeficiency. Paradoxically, IL-4 receptor-deficient, autoreactive CD4 T cells proliferate more extensively than wild-type T cells in immunodeficient hosts, suggesting that the growth-promoting effect of islet IL-4 acts indirectly. These results suggest that IL-4-mediated protection against autoimmunity and diabetes may be outweighed during immunodeficiency by a pathogenic, IL-4-induced expansion of autoreactive T cells.

Key Words: cytokine • autoimmunity • diabetes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-4 is an effective growth factor for resting [1 , 2 ] and activated/memory [3 ] T cells in vitro. It is therefore in some ways paradoxical that IL-4 treatment should also be highly protective in models of autoimmune diseases such as Type 1 diabetes [4 ] and multiple sclerosis (MS) [5 ], which depend on a pathogenic activation and expansion of self-reactive T cells. The expression of IL-4 in islets prevents diabetes in the NOD mouse and induces a functional tolerance to islet antigens [6 ]. Islet IL-4 also prevents lymphocytic choriomeningitis virus (LCMV)-induced diabetes, and inhibits the acquisition of cytotoxicity by CD8 T cells following viral infection by blocking the maturation of antigen-presenting dendritic cells [7 ]. In this model, islet IL-4 promotes a slight increase in the expansion of CD8 T cells specific for the immunodominant epitope of LCMV yet is still able to inhibit cytotoxicity. The growth-promoting effects of IL-4 are therefore not necessarily linked to the development of autoimmunity. However, expression of islet IL-4 in immunodeficient NODScid recipients greatly enhances the expansion of adoptively transferred islet-specific CD4 T cells and accelerates insulitis [8 ]. The ability of IL-4 to affect T cell expansion in vivo, and the contribution of this aspect of IL-4 activity in mediating protection against autoimmunity, are therefore important questions for understanding how IL-4 may be used therapeutically.

The question of whether IL-4 can promote T cell survival in vivo is controversial. Coinjection of IL-4 with staphylococcal enterotoxin A (SEA) inhibits the deletion of Vß3+ cells that occurs following SEA injection alone [3 ], and deprivation of IL-4 and IL-7 enhances the loss of naïve CD4 T cells following thymectomy, suggesting that IL-4 can prevent T cell loss [9 ]. IL-4 promotes the homeostatic proliferation and survival of CD8 T cells in lymphoid organ culture, although it is not required for homeostatic proliferation of CD4 or CD8 T cells in vivo [10 ]. However, there is also evidence that IL-4 can inhibit T cell survival. IL-4-deficient mice have a twofold increase in thymocyte number by 6 weeks of age compared with wild-type [11 ], whereas T cell-specific, transgenic expression of IL-4 reduces thymocyte number dramatically [12 ]. In IL-4-deficient mice on the NOD background, the decay of peripheral T cell numbers following thymectomy is lessened, suggesting that IL-4 inhibits T cell survival in these mice [13 ].

In animal models of diabetes and MS, IL-4 has been shown to improve protection induced by tolerizing DNA vaccination [14 15 16 ]. IL-4 also plays an important role in {alpha}-galactosylceramide-induced protection against diabetes [17 ]. Furthermore, glatiramer acetate (copolymer 1) is approved for the clinical treatment of MS, and its effectiveness is thought to depend on the induction of IL-4-producing Th2 cells [18 ]. However, there are also concerning reports that IL-4 may, under some circumstances, be involved in autoimmune pathogenesis [19 , 20 ]. It is therefore of clinical importance to understand the mechanisms by which IL-4 can protect against autoimmunity and the conditions under which the protective role of IL-4 is compromised.

As IL-4 appears able to both promote and counter T cell survival in vivo, we tested whether the biological effects of this molecule may be dependent on T cell density in the host. We show that the dramatic expansion of autoreactive CD4 T cells induced by islet IL-4 under conditions of severe lymphopenia is inhibited incrementally by the presence of increasing numbers of bystander T cells. Therefore, the ability of IL-4 to promote the expansion of autoreactive CD4 T cells depends on the degree of T cell deficiency. Paradoxically, IL-4 receptor (IL-4R)-deficient, autoreactive CD4 T cells have enhanced proliferation also, suggesting that islet IL-4 may act indirectly to promote proliferation in immunodeficient recipients. These results may explain the unexpected pathogenicity of Th2 cells in immunodeficient hosts [19 ] and suggest that the beneficial effects of IL-4 in protecting against autoimmunity may be outweighed by a pathogenic expansion of autoreactive T cells by IL-4 under conditions of immunodeficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
IL-4NOD [6 ] and IL-4NODScid [8 ] mice, which express IL-4 specifically in their islet ß cells, have been described previously. BDC2.5NOD mice [21 ] were a kind gift of Drs. Diane Mathis and Christophe Benoist (Harvard University, Cambridge, MA, USA), and NODIL-4 knockout (KO) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA; Stock 004222). BALB/c-IL-4ratm1Sz was obtained from Jackson Laboratory (Stock 003514) and backcrossed to NOD mice for nine generations using a speed congenic screening strategy at each generation. N9 mice were NOD-derived at all markers tested across the genome, except for a congenic interval of less than 5 cM flanking the IL-4R-targeted mutation on Chr. 7. The mice were then intercrossed to generate homozygous NOD.IL-4RKO mice. Specific details of the polymorphic markers used for screening are given in Supplemental Figure 1. Inheritance of the IL-4R mutation was tested as described in the Jackson Laboratory PCR protocol. NOD.IL-4RKO mice were then crossed with BDC2.5.NOD mice to generate BDC2.5NOD.IL-4RKO mice expressing a single copy of the BDC2.5 transgene. All strains were maintained under specific, pathogen-free conditions in the rodent-breeding colony at The Scripps Research Institute (La Jolla, CA, USA). Live animal experiments were approved by the Institutional Animal Care and Use Committee and the Animal Research Committee and were conducted in accordance with institutional guidelines for animal care and use.

Adoptive transfer
Adoptive transfer of 20 million CFSE-labeled splenocytes from 5- to 6-week-old BDC2.5NOD or BDC2.5NOD.IL-4RKO donor mice was performed as described previously [8 ]. Approximately 20% splenocytes from BDC2.5NOD mice were islet-specific BDC2.5 T cells. Typically, less than 25% BDC2.5 T cells transferred were CD44hi, and therefore the majority of cells was phenotypically naïve. On Day 4 following transfer, single-cell suspensions from recipient pancreatic lymph nodes (panLN) were prepared for analysis by flow cytometry to detect the CFSE+CD4+Vß4+ BDC2.5 population transferred, as described previously [8 ]. For annexin staining, cells were gated on the live CFSE+CD4+7-amino-actinomycin– population. Flow cytometry was performed using a FACSCaliber cytometer and CellQuest software or digital LSR II with DiVa and FlowJo software. All average values are shown ± SEM, and an unpaired Student’s t-test (two-tailed) was used where indicated to test statistical significance. Bystander cells were whole splenocytes isolated from 8-week-old NOD mice, and 0, 20, or 100 million cells were injected into Scid recipients at the same time as the CFSE-labeled BDC2.5 splenocytes.

Immunohistochemistry
The pancreas of recipient mice was quick-frozen in OCT medium at Day 4 following transfer, and 4 µm sections from three levels, each level separated by at least 120 µm, were stained with anti-insulin (Dako, Carpinteria, CA, USA; 1/400) and Texas Red-conjugated antiguinea pig secondary antibody (Vector Laboratories, Burlingame, CA, USA; 1/200). 4',6-Diamidino-2-phenylindole (DAPI) nuclear dye was included in the mounting media to visualize nuclei (blue), and the transferred cells were visible as a result of the CFSE label (green). Images were taken using a Bio-Rad (Zeiss) Radiance 2100 rainbow laser-scanning confocal microscope and 40x oil objective lens using Bio-Rad LaserSharp (v3.2) software. ImageJ [National Institutes of Health (NIH), Bethesda, MD, USA] was used for image analysis. The endogenous and CFSE+ infiltration were scored separately for each islet, and islets were scored as having no insulitis, peri-insulitis, insulitis, or severe insulitis, and differences in the distributions were determined using a {chi}2 test. Sections from a total of two IL-4NOD and two NOD pancreata, from two independent experiments, were examined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4 promotes the expansion of autoreactive CD4 T cells only under conditions of lymphopenia
We have shown previously that the presence of islet IL-4 enhances the expansion of islet-specific, autoreactive CD4 T cells dramatically when adoptively transferred into lymphopenic (Scid) recipients [8 ]. To test our hypothesis that immunodeficiency is important for the increased expansion of autoreactive T cells by IL-4, we performed adoptive transfer experiments into islet-IL-4 NOD mice, which contain their normal complement of lymphocytes. Islet-reactive CD4 T cells from BDC2.5NOD mice [21 ] were CFSE-labeled and adoptively transferred into wild-type or islet-IL-4 recipients on the NOD and NODScid backgrounds in parallel. At Day 4 following transfer, the number of CFSE+CD4+ BDC2.5 T cells in the panLN was determined (Fig. 1A ). Consistent with our results reported previously, in control, lymphopenic Scid recipients, islet IL-4 increased recovery of the autoreactive BDC2.5 T cell population (6.2-fold, P=0.02, n=3) [8 ]. However, on the immunocompetent NOD background, the presence of IL-4 did not increase the recovery of BDC2.5 cells, and in fact, their number was reduced slightly in IL-4NOD compared with NOD mice (1.7-fold, n=3), although the difference did not reach statistical significance. To test whether the BDC2.5 T cells in IL-4NOD mice undergo enhanced expansion but that an increased rate of apoptosis counters accumulation, we examined the CFSE profile and annexin staining of these cells. However, no significant difference in the CFSE division profile (Fig. 1B and 1C) or annexin staining (Fig. 1D) of the BDC2.5 T cell population was observed between IL-4NOD and NOD recipients. In IL-4NODScid recipients, the BDC2.5 cells were seen to undergo increased proliferation and reduced annexin-V staining compared with NODScid recipients, as reported previously [8 ]. IL-4 therefore only promotes the expansion of autoreactive CD4 T cells under conditions of lymphopenia and does not impact autoreactive CD4 T cell expansion significantly in normal NOD mice. We also performed adoptive transfers using IL-4-deficient NOD recipients and observed an equivalent number of BDC2.5 T cells to that in wild-type NOD recipients (Fig. 1E) and a comparable CFSE profile (Fig. 1F) . Autoreactive CD4 T cell expansion is therefore unaffected by IL-4 deficiency and the presence of islet IL-4 in immunocompetent mice.


Figure 1
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  		Figure 1. Autoreactive CD4 T cell expansion is only increased by IL-4 under conditions of lymphopenia. Adoptively transferred BDC2.5 T cells were recovered from the panLN at Day 4 following transfer. Recipients were wild-type (wt) or expressed islet IL-4 and on immunodeficient NODScid and normal NOD backgrounds. The number (A), CFSE division profile (B, C), and annexin V staining (D) of BDC2.5 T cells are shown. n = 3 for each recipient strain, representative of three independent experiments. BDC2.5 T cells were also transferred to NOD (n=9) and IL-4-deficient NOD (n=7) mice, and the recovery (E) and CFSE prolife (F) are shown. Data pooled from two experiments showing the same result. The average value ± SEM is shown in each case.

 
Reduced islet infiltration by adoptively transferred, autoreactive CD4 T cells in NOD mice expressing islet IL-4
In NODScid recipients, the increased expansion of autoreactive CD4 T cells in mice expressing islet IL-4 was associated with an acceleration of insulitis [8 ]. To test whether the lack of this expansion in IL-4NOD recipients is associated with protection against insulitis, we examined the islets of IL-4NOD and NOD recipients for the presence of transferred BDC2.5 cells. As shown in Figure 2A , CFSE-labeled BDC2.5 splenocytes can be found in the islets of NOD and IL-4NOD recipients at Day 4 post-transfer. Insulitis scoring (Fig. 2B) was performed for injected (CFSE-positive) and endogenous (areas of dense nuclear stain) cells and was found to be significantly less severe in IL-4NOD recipients (P<0.05 in both cases). Islet IL-4 therefore protects against islet infiltration by endogenous and injected BDC2.5 T cells. This also suggests that the slight reduction in the number of BDC2.5 T cells in the panLN of IL-4NOD recipients compared with NOD is unlikely to be a result of increased homing of activated cells to the pancreas. The number of CFSE-positive cells within each islet was observed to correlate with the degree of endogenous infiltration, and there was no difference in the distribution of insulitis scoring between endogenous and BDC2.5 cells in either strain. Therefore, in immunosufficient NOD mice, where endogenous islet inflammation is already present, the degree of pre-existing inflammation may be the primary determinant of BDC2.5 T cell islet recruitment. Hence, on the NOD background islet, IL-4 does not significantly affect expansion in the panLN, but the extent of islet infiltration is reduced, possibly as a result of the reduction in endogenous insulitis in this strain.


Figure 2
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  		Figure 2. Infiltration of injected, autoreactive T cells corresponds with the degree of endogenous infiltration in NOD and IL-4NOD recipients. Sections through the pancreas of NOD (i, ii) and IL-4NOD (iii, iv) recipient mice at Day 4 following transfer of CFSE-labeled BDC2.5 splenocytes (green) are shown stained with antibodies to insulin (red) and with DAPI (blue) as a nuclear dye (A). Representative islets with mild (i, iii) and more extensive (ii, iv) areas of infiltration are shown. Insulitis scoring for endogenous and injected cells is shown (B), and the values used to perform {chi}2 analysis are given (C).

 
Cotransfer of bystander cells decreases the recovery of autoreactive CD4 T cells in recipients expressing islet IL-4 and increases their recovery in wild-type mice
The inability of IL-4 to enhance autoreactive T cell expansion except in lymphopenic Scid mice suggests that the growth-promoting activity of IL-4 occurs only in the absence of adequate T cell numbers. However, homeostatic proliferation in congenitally T cell-deficient hosts occurs more rapidly than in lymphocyte-depleted, normal hosts, and one suggestion is that this is driven by the immunogenic effect of increased levels of foreign antigens [22 ]. The presence of islet IL-4 could potentially be enhancing this immunogenic effect rather than reflecting T cell deficiency, or compounding another effect that is specific to congenitally immunodeficient mice. We therefore wanted to test whether we could inhibit the growth-promoting effect of IL-4 on BDC2.5 T cells in Scid recipients by cotransferring increasing numbers of bystander cells.

We cotransferred 0, 20, or 100 million unlabeled splenocytes from NOD mice, in addition to the 20 million CFSE-labeled BDC2.5 splenocytes, into Scid recipients. Transfer of increasing bystander cell numbers increased the size of the bystander CD4 T cell population in wild-type and islet IL-4-expressing recipients (data not shown). Cotransferring an increasing number of bystander cells into wild-type Scid recipients increased the recovery of BDC2.5 cells in the panLN (Fig. 3 ). It therefore appears that the presence of bystander cells increases the percentage of injected cells recovered. However, in contrast, the recovery of BDC2.5 cells from IL-4NODScid recipients was reduced by the cotransfer of bystander cells and decreased incrementally as the number of bystander cells was increased. Hence, there is a clearly contrasting positive and negative correlation between bystander cell number and BDC2.5 recovery in wild-type and islet-IL-4 recipients, respectively, which is evident despite the inherent problem of variability when determining absolute cell counts in adoptively transferred Scid recipients. This suggests that the increasing, growth-promoting effect of IL-4 on autoreactive CD4 T cells under conditions of immunodeficiency outweighs the loss of the positive effects of bystander cells on T cell recovery. These experiments also demonstrate that the ability of IL-4 to promote autoreactive CD4 T cell expansion is critically dependent on the absence of sufficient T cell numbers.


Figure 3
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  		Figure 3. Bystander cells decrease the recovery of autoreactive CD4 T cells in the presence of islet IL-4 but increase their recovery in wild-type mice. CFSE-labeled BDC2.5 splenocytes were coinjected with the indicated number of unlabeled bystander NOD splenocytes, and the number of CFSE+CD4+ BDC2.5 T cells recovered from the panLN at Day 4 was determined. Plots show average BDC2.5 cell number recovered ± SEM. (A) Wild-type and islet IL-4 Scid recipients were coinjected with 0, 20, or 100 million bystander cells, and (B) wild-type and islet IL-4 Scid recipients were coinjected with 20 or 100 million bystander cells, and BDC2.5 cells were also transferred into NOD and IL-4NOD recipients in parallel. n = 3–5 each strain in each experiment.

 
The accumulation of proliferating cells by IL-4 is gradually inhibited by increasing the number of bystander cells
The CFSE dilution profile of autoreactive CD4 T cells transferred to Scid mice expressing islet IL-4 shows a pronounced accumulation of proliferating cells at Day 4 following transfer (Fig. 1C and ref. [8 ]). To test whether bystander cells inhibit this accumulation of dividing cells, we compared the CFSE profile of BDC2.5 T cells cotransferred into Scid recipients with titrated numbers of bystander cells (Fig. 4A ). Somewhat surprisingly, the CFSE profile of transferred BDC2.5 T cells in wild-type Scid recipients was unaffected by the number of bystander cells present. We quantified the percentage of BDC2.5 T cells, which have not undergone cell division (Fig. 4B and 4C) and found that the percent of undivided cells was not different in wild-type Scid mice, with and without cotransferred bystander cells, and in NOD mice. The proliferation of BDC2.5 T cells that we observe in these experiments is therefore unaffected by the degree of lymphopenia in the recipient. In contrast, the accumulation of proliferating cells by IL-4 was gradually inhibited by the transfer of an increasing number of bystander cells, demonstrating that this accumulation is dependent directly on T cell deficiency. The CFSE profile of BDC2.5 T cells in IL-4NODScid recipients cotransferred with 100 million bystander cells was equivalent to that seen in wild-type recipients. The number of CD4 T cells present in the panLN of Scid mice reconstituted with 100 million cells is still less than 10% of the normal complement of CD4 T cells in full NOD mice in the presence of islet IL-4 or not (data not shown). The growth-promoting effects of IL-4 are therefore inhibited by the presence of T cell numbers well below those of a normal NOD mouse. IL-4 therefore differentially affects the expansion of autoreactive CD4 T cells according to the degree of lymphopenia, and under conditions of severe lymphopenia, IL-4 is able to dramatically promote autoreactive CD4 T cell expansion.


Figure 4
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  		Figure 4. The accumulation of proliferating cells in the presence of islet IL-4 is inhibited gradually by increasing the number of bystander cells. (A) The CFSE profile for BDC2.5 T cells in the experiment in Figure 3A is shown, and (B and C) the average percent cells that have not undergone division for the experiments in Figure 3A and 3B , respectively, are shown. Cells gated on CFSE+CD4+ population in each case.

 
Gating on CFSE+ cells excludes primarily endogenous T cells in NODScid recipients
To compare the CFSE profiles in NOD mice, it was necessary to gate on the CFSE-positive cells to distinguish the injected BDC2.5 T cells from endogenous CD4+Vß4+ T cells, which are present at high frequency. The same gate was used for NODScid recipients. However, a population of CFSE– cells in NODScid recipients is excluded by this gate, and it is not clear if these are highly proliferating cells that are being excluded from the analysis or endogenous cells that can arise in "leaky" Scid mice. We therefore used an antibody raised specifically against the BDC2.5 TCR to test whether these cells are injected or endogenous cells [23 ]. In control, uninjected NOD mice, the endogenous BDC2.5 T cell population stained by the antibody was observed to make up 0.8% of the CD4 T cell pool (Fig. 5A ), comparable with results reported previously [23 ]. As shown in Figure 5B , the majority of CFSE+CD4+ cells in NOD and Scid recipients stains positively with the anti-BDC antibody. However, the CD4+CFSE– cells in Scid mice, which have been excluded by the CFSE gate, are largely endogenous, non-BDC2.5 T cells rather than injected cells that have proliferated extensively and lost the CFSE signal. Consistent with this, a similar population can be observed in control, uninjected mice. Therefore, although some of the highly proliferating BDC2.5 T cells in IL-4NODScid mice are likely to be lost with the CFSE gate, this should not affect the conclusion that expansion is greater in this strain, and comparisons between BDC2.5 T cell proliferation in NOD and Scid recipients are likely to be impacted only mildly.


Figure 5
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  		Figure 5. CD4+CFSE– cells in NODScid recipients are largely endogenous T cells. Lymphoid organs from NOD and NODScid mice, unmanipulated or injected with CFSE-labeled BDC2.5 splenocytes (Day 4), were stained with antibodies to CD4 and Vß4 and an antibody specific for the BDC2.5 clonotype [23 ]. (A) Staining of CD4+ cells in unmanipulated NOD mice with the anti-BDC antibody and the relevant isotype control and (B) staining of CFSE+ and CFSE– poulations of CD4+ T cells with the anti-BDC antibody. Boxed region in the anti-BDC dot plots indicates clonotype high cells.

 
IL-4R deficiency on BDC2.5 T cells promotes their expansion in immunodeficient NODScid mice
To test whether the growth-promoting effect of IL-4 under conditions of immunodeficiency is directly a result of signaling through IL-4R on the BDC2.5 T cells, or an indirect effect via another cell type, we adoptively transferred CFSE-labeled BDC2.5.IL-4RKO T cells into NODScid recipients. The recovery of IL-4R-deficient BDC2.5 T cells from the panLN of Scid mice at Day 4 following transfer was not different than that of wild-type BDC2.5 T cells (Fig. 6A ). However, the CFSE profile demonstrates that BDC2.5.IL-4RKO T cells exhibit a much greater accumulation of proliferating cells in the panLN than wild-type BDC2.5 T cells (Fig. 6B and 6C) . This suggests that the presence of IL-4R on autoreactive CD4 T cells normally acts to inhibit proliferation in immunodeficient mice. Therefore, although islet expression of IL-4 promotes BDC2.5 T cell expansion in the panLN of immunodeficient mice, when BDC2.5 T cells lack IL-4R, this also results paradoxically in enhanced panLN proliferation. This suggests that either there is a dose-dependence of the effect of IL-4 on autoreactive T cell expansion or that the growth-enhancing effect of islet IL-4 on autoreactive T cells during immunodeficiency may act indirectly through another cell type.


Figure 6
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  		Figure 6. IL-4R deficiency in BDC2.5 T cells increases autoantigen-driven proliferation under conditions of immunodeficiency. panLN and ingLN cells from NODScid mice injected with CFSE-labeled splenocytes from BDC2.5 and BDC2.5IL-4RKO mice, and also NOD and young NOD (NODy; at 3.5 weeks of age as recipients) mice injected with the same BDC2.5 cells in parallel, at Day 4 following transfer. The number (A), percent undivided cells (B), and representative CFSE profiles (C and D) are shown. n = 3 for each strain/cell type. PanLN, pancreatic lymph node; IngLN, inguinal lymph node.

 
We also performed additional controls to test the dependence of IL-4R deficiency-induced proliferation on the presence of autoantigen. In the ingLN of NODScid mice, where islet antigen is not present, proliferation of BDC2.5.IL-4RKO cells was not increased compared with wild-type BDC2.5 cells (Fig. 6D) . This suggests that, as in IL-4NODScid mice [8 ], the increased proliferation of BDC2.5.IL-4RKO T cells in panLN is dependent on the presence of autoantigen. We also transferred BDC2.5 T cells into immunosufficient NOD recipients in parallel. As an additional control, we used NOD mice at 3.5 weeks of age as recipients (NODy) as this is the approximate age at which loss of tolerance to islet autoantigens in the panLN is thought to be initiated [24 ]. In these mice, the expansion of BDC2.5 T cells was seen to be reduced compared with the usual 6- to 7-week-old NOD recipients, suggesting that it is the presence of large amounts of autoantigen resulting from T cell-dependent islet damage that is driving the high degree of proliferation in the panLN of immunosufficient mice.

Figure 6 also shows that BDC2.5 T cells in the ingLN of NODScid mice have undergone one to two rounds of division, and little proliferation is evident in the ingLN of NOD mice. Lymphopenia-induced proliferation of BDC2.5 T cells is therefore evident in the ingLN of NODScid mice. However, the effect of the Scid environment in increasing cell division in panLN BDC2.5 T cells was again less evident than expected. Proliferation of islet-reactive CD4 T cells in the panLN is clearly autoantigen-driven, and particularly, as NOD mice are mildly lymphopenic [25 ], this may represent a maximal rate of proliferation in NOD or NODScid mice, except where increased levels of exogenous cytokines are present. Yet, the recovery of injected BDC2.5 T cells from the ingLN of NODScid recipients is reduced even more strikingly compared with NOD recipients than in the panLN, supporting the idea that although proliferation may increase in Scid recipients, the recovery of naïve cells recruited to LN is markedly reduced.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These experiments demonstrate that the ability of islet IL-4 to promote the accumulation of proliferating cells is dependent on conditions of severe immunodeficiency. However, IL-4R-deficient, autoreactive CD4 T cells were found to proliferate more extensively in immunodeficient recipients, suggesting that islet IL-4 perhaps acts indirectly to promote autoreactive T cell expansion. The opposing effects of IL-4 under immunosufficient and immunodeficient conditions may therefore reflect the predominance of IL-4 acting directly or indirectly on T cells. The results also suggest that the presence of the IL-4Rß chain limits the proliferation of autoreactive CD4 T cells in immunodeficient hosts. Further experiments will be required to understand the mechanism that underlies this, but one possibility is that responsiveness to another {gamma}c cytokine is enhanced in T cells lacking IL-4Rß.

It is important to understand the growth-promoting and homeostatic effects of cytokines on autoreactive T cells in an in vivo context. The NOD mouse was itself recently shown to be mildly lymphopenic [25 ], and homeostatic expansion or hypersensitivity to homeostatic cytokines can drive self-reactive T cells to cause autoimmunity [25 , 26 ]. The growth-promoting effects of IL-4 in an immunodeficient context are associated with an acceleration of insulitis [8 ]. Our results suggest that direct and indirect effects of IL-4, as well as the degree of T cell immunodeficiency, can differentially affect the outcome of IL-4 exposure on autoreactive CD4 T cell expansion. Therefore, although IL-4 is profoundly protective against diabetes under conditions of immunosufficiency and is able to induce tolerance via its effects on APC, our results suggest that in the context of immunodeficiency, the growth-promoting effects of IL-4 on autoreactive T cells may predominate and overcome protection and even accelerate disease.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH Grant DK054063 and American Diabetes Association Mentor-Based Postdoctoral Fellowship Award #7-05-MN-53. We thank Cecile King for useful, general discussions, Jared Purton and members of the Sarvetnick lab for comments about the manuscript, and the TSRI Flow Cytometry Core Facility.

Received April 5, 2006; accepted November 16, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Boise, L. H., Minn, A. J., June, C. H., Lindsten, T., Thompson, C. B. (1995) Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division Proc. Natl. Acad. Sci. USA 92,5491-5495[Abstract/Free Full Text]
  2. Vella, A., Teague, T. K., Ihle, J., Kappler, J., Marrack, P. (1997) Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: STAT6 is probably not required for the effect of IL-4 J. Exp. Med. 186,325-330[Abstract/Free Full Text]
  3. Vella, A. T., Dow, S., Potter, T. A., Kappler, J., Marrack, P. (1998) Cytokine-induced survival of activated T cells in vitro and in vivo Proc. Natl. Acad. Sci. USA 95,3810-3815[Abstract/Free Full Text]
  4. Rapoport, M. J., Jaramillo, A., Zipris, D., Lazarus, A. H., Serreze, D. V., Leiter, E. H., Cyopick, P., Danska, J. S., Delovitch, T. L. (1993) Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice J. Exp. Med. 178,87-99[Abstract/Free Full Text]
  5. Shaw, M. K., Lorens, J. B., Dhawan, A., DalCanto, R., Tse, H. Y., Tran, A. B., Bonpane, C., Eswaran, S. L., Brocke, S., Sarvetnick, N., Steinman, L., Nolan, G. P., Fathman, C. G. (1997) Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis J. Exp. Med. 185,1711-1714[Abstract/Free Full Text]
  6. Mueller, R., Krahl, T., Sarvetnick, N. (1996) Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice J. Exp. Med. 184,1093-1099[Abstract/Free Full Text]
  7. King, C., Mueller Hoenger, R., Malo Cleary, M., Murali-Krishna, K., Ahmed, R., King, E., Sarvetnick, N. (2001) Interleukin-4 acts at the locus of the antigen-presenting dendritic cell to counter-regulate cytotoxic CD8+ T-cell responses Nat. Med. 7,206-214[CrossRef][Medline]
  8. Hill, N. J., Van Gunst, K., Sarvetnick, N. (2003) Th1 and Th2 pancreatic inflammation differentially affects homing of islet-reactive CD4 cells in nonobese diabetic mice J. Immunol. 170,1649-1658[Abstract/Free Full Text]
  9. Boursalian, T. E., Bottomly, K. (1999) Survival of naive CD4 T cells: roles of restricting versus selecting MHC class II and cytokine milieu J. Immunol. 162,3795-3801[Abstract/Free Full Text]
  10. Tan, J. T., Dudl, E., LeRoy, E., Murray, R., Sprent, J., Weinberg, K. I., Surh, C. D. (2001) IL-7 is critical for homeostatic proliferation and survival of naive T cells Proc. Natl. Acad. Sci. USA 98,8732-8737[Abstract/Free Full Text]
  11. Kuhn, R., Rajewsky, K., Muller, W. (1991) Generation and analysis of interleukin-4-deficient mice Science 254,707-710[Abstract/Free Full Text]
  12. Lewis, D. B., Yu, C. C., Forbush, K. A., Carpenter, J., Sato, T. A., Grossman, A., Liggitt, D. H., Perlmutter, R. M. (1991) Interleukin 4 expressed in situ selectively alters thymocyte development J. Exp. Med. 173,89-100[Abstract/Free Full Text]
  13. Vivien, L., Benoist, C., Mathis, D. (2001) T lymphocytes need IL-7 but not IL-4 or IL-6 to survive in vivo Int. Immunol. 13,763-768[Abstract/Free Full Text]
  14. Tisch, R., Wang, B., Weaver, D. J., Liu, B., Bui, T., Arthos, J., Serreze, D. V. (2001) Antigen-specific mediated suppression of ß cell autoimmunity by plasmid DNA vaccination J. Immunol. 166,2122-2132[Abstract/Free Full Text]
  15. Wolfe, T., Bot, A., Hughes, A., Mohrle, U., Rodrigo, E., Jaume, J. C., Baekkeskov, S., von Herrath, M. (2002) Endogenous expression levels of autoantigens influence success or failure of DNA immunizations to prevent type 1 diabetes: addition of IL-4 increases safety Eur. J. Immunol. 32,113-121[CrossRef][Medline]
  16. Garren, H., Ruiz, P. J., Watkins, T. A., Fontoura, P., Nguyen, L. T., Estline, E. R., Hirschberg, D. L., Steinman, L. (2001) Combination of gene delivery and DNA vaccination to protect from and reverse Th1 autoimmune disease via deviation to the Th2 pathway Immunity 15,15-22[CrossRef][Medline]
  17. Mi, Q. S., Ly, D., Zucker, P., McGarry, M., Delovitch, T. L. (2004) Interleukin-4 but not interleukin-10 protects against spontaneous and recurrent type 1 diabetes by activated CD1d-restricted invariant natural killer T-cells Diabetes 53,1303-1310[Abstract/Free Full Text]
  18. Aharoni, R., Meshorer, A., Sela, M., Arnon, R. (2002) Oral treatment of mice with copolymer 1 (glatiramer acetate) results in the accumulation of specific Th2 cells in the central nervous system J. Neuroimmunol. 126,58-68[CrossRef][Medline]
  19. Lafaille, J. J., Keere, F. V., Hsu, A. L., Baron, J. L., Haas, W., Raine, C. S., Tonegawa, S. (1997) Myelin basic protein-specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather than protect them from the disease J. Exp. Med. 186,307-312[Abstract/Free Full Text]
  20. Ohmura, K., Nguyen, L. T., Locksley, R. M., Mathis, D., Benoist, C. (2005) Interleukin-4 can be a key positive regulator of inflammatory arthritis Arthritis Rheum. 52,1866-1875[CrossRef][Medline]
  21. Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. (1993) Following a diabetogenic T cell from genesis through pathogenesis Cell 74,1089-1100[CrossRef][Medline]
  22. Surh, C. D., Sprent, J. (2005) Regulation of mature T cell homeostasis Semin. Immunol. 17,183-191[CrossRef][Medline]
  23. Kanagawa, O., Militech, A., Vaupel, B. A. (2002) Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice J. Immunol. 168,6159-6164[Abstract/Free Full Text]
  24. Hoglund, P., Mintern, J., Waltzinger, C., Heath, W., Benoist, C., Mathis, D. (1999) Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes J. Exp. Med. 189,331-339[Abstract/Free Full Text]
  25. King, C., Ilic, A., Koelsch, K., Sarvetnick, N. (2004) Homeostatic expansion of T cells during immune insufficiency generates autoimmunity Cell 117,265-277[CrossRef][Medline]
  26. Davey, G. M., Starr, R., Cornish, A. L., Burghardt, J. T., Alexander, W. S., Carbone, F. R., Surh, C. D., Heath, W. R. (2005) SOCS-1 regulates IL-15-driven homeostatic proliferation of antigen-naive CD8 T cells, limiting their autoimmune potential J. Exp. Med. 202,1099-1108[Abstract/Free Full Text]




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