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The Journal of Immunology, 2007, 179, 2844 -2850
Copyright © 2007 by The American Association of Immunologists, Inc.
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Targeting T Cell-Specific Costimulators and Growth Factors in a Model of Autoimmune Hemolytic Anemia1

Katrina K. Hoyer, Kristen Wolslegel, Hans Dooms and Abul K. Abbas2

Department of Pathology, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although it is established that failure of regulatory mechanisms underlies many autoimmune diseases, the stimuli that activate autoreactive lymphocytes remain poorly understood. Defining these stimuli will lead to therapeutic strategies for autoimmune diseases. IL-2-deficient mice develop spontaneous autoimmunity, because of a deficiency of regulatory T cells, and on the BALB/c background, they rapidly die from autoimmune hemolytic anemia. To define the importance of costimulatory pathways in various components of this autoimmune disorder, we first intercrossed IL-2-deficient mice with mice lacking CD28 or CD40L. Elimination of CD28 reduced the activation of autoreactive T cells and lymphoproliferation as well as production of autoantibodies, whereas elimination of CD40L reduced autoantibody production without affecting T cell expansion and accumulation. To examine the role of IL-7, we blocked IL-7R signaling with neutralizing Abs. This treatment inhibited the production of autoantibodies and the development of autoimmune hemolytic anemia. Together, these data indicate that specific costimulatory and cytokine signals are critical for the spontaneous autoantibody-mediated disease that develops in IL-2-deficient mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Autoimmune hemolytic anemia (AIHA)3 is characterized by the production of Abs directed against self RBC. Given the frequent association between AIHA and other autoimmune disorders, generalized immune dysfunction most likely plays a role in the disease process. Under normal conditions, self-reactive lymphocytes are normally killed, inactivated, or suppressed by regulatory T cells, resulting in unresponsiveness to self Ags (1, 2, 3, 4). Disruption of these control mechanisms results in the survival and pathologic activation of self-reactive lymphocytes. It is unclear how self-reactive lymphocytes are spontaneously activated in the absence of overt infection or other stimuli, leading to autoimmune disease. A clearer definition of the costimulatory and cytokine pathways leading to spontaneous autoimmune disease may point to new therapies for these disorders, as Ab blockade of cytokine signaling has done for Crohn’s disease and rheumatoid arthritis (5, 6). Conventional dogma states that costimulatory signals (especially those provided by the B7:CD28 pathway) are triggered by infectious agents. Blocking these signals can promote tolerance to self Ags (reviewed in Ref. 7). Costimulatory signals have also been implicated in the development of autoimmunity. However, many of the studies supporting this conclusion have used experimental models of overt immunization with self Ags in adjuvant to activate autoreactive lymphocytes. Such immunization is known to induce innate immune responses, so it is not surprising that in such models the requirements for T cell activation resemble those for normal T cell responses. Much less is known about the role of costimulatory and cytokine signals in the spontaneous activation of self-reactive lymphocytes. Studies of spontaneous diabetes in the NOD mouse, for example, have shown that removal of B7:CD28 costimulation exacerbates disease by reducing the development and survival of regulatory T cells (8). Thus, in this setting, the role of well-known costimulatory pathway is entirely unexpected.

We used a mouse model of spontaneous, acute systemic autoimmunity induced by elimination of regulatory T cells to define the stimuli that are required for the development of autoimmune disease. IL-2-knockout (KO) mice on the BALB/c background develop a systemic autoimmune disease, dying between 3 and 5 wk from complications of AIHA (9). The principal defects in these mice are a lack of regulatory T lymphocytes (Tregs) leading to a breakdown of self-tolerance and failure of T cell homeostasis, resulting in uncontrolled activation and proliferation of CD4+ T cells (10, 11). It has been shown that AIHA progression in these animals is mediated by autoantibodies and is dependent on abnormal Th cell activity (9, 12).

In this study, we set out to address two questions, as follows: 1) what aspects of the autoimmune phenotype are influenced by specific lymphocyte activation pathways, and 2) can we identify the cytokines that compensate for the absence of IL-2 in the activation of self-reactive lymphocytes? Our results demonstrate that the removal of either CD28 or CD40L costimulation or IL-7 signaling delays or eliminates AIHA onset, and suggest that blockade of these signals is a potential entry point for therapeutic strategies for an acute autoantibody-mediated disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

For these experiments, mice lacking IL-2, CD28, CD40L, IL-2/CD28, IL-2/CD40L, or TCR{alpha} were used on the BALB/c background. All mice were bred and maintained in our specific pathogen-free facility at the Animal Barrier Facility in accordance with the guidelines of the Laboratory Animal Resource Center of the University of California.

Lymphocyte isolation

Lymph nodes and spleens were pressed through a nylon mesh filter, RBC were hypotonically lysed, and then lymphocytes were washed and resuspended in PBS with 1% FBS.

Abs and flow cytometry

Splenocytes and lymphocytes were stained with FITC-, PE-, PerCP-, or allophycocyanin-conjugated Abs following Fc block (anti-CD16/CD32). All Abs were purchased from BD Pharmingen, unless otherwise noted. Flow cytometry was performed on a FACSCalibur (BD Biosciences), and data were analyzed using FCS Express (DeNovo Software).

Complete blood counts

Cardiac punctures were performed immediately following cervical dislocation, and blood was drawn into heparinized microhematocrit tubes. Complete blood counts (including erythrocyte and white blood cell counts, hematocrit percentages, and hemoglobin values) were then evaluated using a Hematovet 950.

Cell purification and adoptive transfers

CD4+CD25– cells were purified from lymph nodes and spleens to 99% purity using a MoFlo cell sorter (DakoCytomation). Purified cells were then adoptively transferred by tail vein injection into recipient mice (5 x 106 cells/mouse).

In vivo cytokine depletion

Rat anti-murine IL-7R{alpha} mAbs were purified from the supernatant of hybridoma cell lines (clone A7R34). A total of 20 µg of anti-IL-7R{alpha}-directed Abs or control Ab was injected i.p./g body weight (500 µg/adult mouse) three times per week beginning on or before day 9. Control rat IgG was obtained from Jackson ImmunoResearch Laboratories.

ELISAs

Ig ELISAs were performed by standard methods. Briefly, 96-well microtiter plates were incubated overnight at 4°C with 2 µg/ml anti-mouse Ig (H & L) Ab (BD Pharmingen) in PBS. The plates were washed three times with PBS/0.5% BSA/0.1% Tween 20 and blocked for 1 h, and then samples of mouse serum were added in duplicate at increasing serial dilutions. After 2 h, the plates were washed, and alkaline phosphatase-linked anti-IgM or anti-IgG2a Ab was added for 1 h. Finally, wells were washed and incubated with p-nitrophenol phosphate substrate (Sigma-Aldrich), and absorbance was determined with an ELISA plate reader (Molecular Devices) at 405 nm. The Ig concentrations were calculated by comparison against a standard curve of serially diluted IgM or IgG2a.

RBC Ab detection

Serum erythrocyte Ab levels were detected using flow cytometry similar to what has been previously described (13). Erythrocytes were freshly isolated from young wild-type or IL-2-KO BALB/c mice by terminal bleed and washed three times in cold PBS. For indirect staining, mouse serum was serial diluted in PBS-1% BSA, and 100 µl incubated with 10 µl of 1% wild-type erythrocytes for 45 min on ice. For direct staining, no serum was added. Cells were then washed two times and incubated with either anti-murine IgM-FITC (1/300 dilution; on ice), IgG-FITC (1/50 dilution; at 37°C; Jackson ImmunoResearch Laboratories), or rat anti-murine {kappa}-biotin (Zymed Laboratories) at a 1/200 dilution, followed by incubation with strepavidin-FITC at a 1/200 dilution. The percentage of erythrocytes bound by Ab was determined by flow cytometry.

Statistics

Statistical differences between experimental groups were determined by unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BALB/c IL-2-KO mouse model of autoimmune anemia and lymphoproliferative disease

In agreement with published literature, IL-2-KO BALB/c mice in use by our laboratory developed AIHA at 19–27 days of age, as demonstrated by hematocrit percentages relative to wild-type mice (Fig. 1A). Anti-erythrocyte Abs of IgG and IgM isotypes were bound to the erythrocytes from IL-2-KO mice when the mice became visibly ill with anemia (Fig. 1B). The serum also contained anti-erythrocyte Abs (Fig. 1, C and D). No anti-erythrocyte Abs were found in wild-type serum or bound to erythrocytes in wild-type mice. In addition, IL-2-KO mice developed a lymphoproliferative disorder with increased lymph node (Fig. 1E) and splenic cell numbers (data not shown) that correlated with an accumulation of CD4+ and CD8+ T cells and B220+ B cells (described in Figs. 4A and 7). Surface CD44 and CD69 expression was increased, whereas CD62L expression was decreased in IL-2-KO CD4+ T cells (Fig. 1F and data not shown), indicating that the expanded T cell population displayed an activated phenotype. The acute nature of this autoimmune disease and the availability of quantitative assays to measure disease severity make this model an attractive one for testing potential therapeutic strategies.


Figure 1
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  		FIGURE 1. BALB/c IL-2-deficient mice develop autoimmune anemia and lymphoproliferative disease. A, Hematocrit percentages were evaluated at <5 wk. B, Erythrocytes from wild-type (WT; filled histogram) and IL-2-KO (open histograms) were stained with anti-mouse IgM FITC and anti-mouse IgG FITC to measure the level of bound anti-erythrocyte Abs. C and D, Serum anti-erythrocyte Abs were measured from WT and IL-2-KO mice at <5 wk using fresh WT erythrocytes. D, Each line represents serum from one IL-2-KO mouse at <5 wk of age. WT serum had no detectable binding at a 1/50 dilution. E, Lymphocytes from 3- to 5-wk-old mice were stained and analyzed by flow cytometry. Bar graphs indicate the total number of cells (x10–6) in the lymph node. F, CD44 and CD62L activation marker profiles of WT and IL-2-KO CD4+ T cells were examined by flow cytometric analysis. Data are representative from at least four mice per genotype.

 

Figure 4
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  		FIGURE 4. Eliminating CD28, but not CD40L, delays the lymphoproliferative disorder in IL-2-deficient mice. Lymphocytes from 3- to 5-wk-old (A) or 3-mo-old (B) mice were analyzed by flow cytometry. Bar graphs indicate the number of cells (x10–6) of each population in the lymph node and the percentage of CD4+ lymph node T cells expressing the activation marker CD69 or CD44. B, IL-2-KO cell numbers and activation percentages are from 19- to 25-day-old mice. Bars represent the means of six mice per group, and brackets show SDs.

 

Figure 7
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  		FIGURE 7. IL-7R{alpha}-blocking Ab delays lymphoproliferative disorder in IL-2-deficient mice. Total lymph node cell numbers (A), percentage of CD4+CD44high cells (B), and B cell numbers in the lymph node (C) were analyzed by flow cytometry. An unpaired Student’s t test (*, indicates p < 0.05) was used to determine statistical differences between samples with and without IL-7R Ab treatment. Data from four mice collected during two experiments.

 
IL-2-KO CD4+ T cells are capable of transferring autoimmune anemia

Early work using IL-2-KO mice crossed with Rag-2-KO and JH-KO mice established that AIHA can be prevented or delayed by elimination of B or T cells (14). However, it remains unclear whether transferring T lymphocytes to unaffected animals confers disease, thereby establishing these cells as independently sufficient for disease propagation in the presence of normal B lymphocytes. This is an important question if one is to test treatment strategies that specifically target T cells. Therefore, we evaluated the contribution of IL-2-KO CD4+ T lymphocytes to the progression of lymphoproliferation and AIHA. A total of 5 x 106 CD4+CD25– T cells from IL-2-KO or wild-type mice was purified and transferred i.v. into TCR{alpha}-KO recipient mice, which lack T cells, but have normal B cells. Recipient mice became visibly ill (with anemia, lethargy, and hunched or wasted appearance) within 4–6 wk after adoptive transfer of IL-2-KO CD4+CD25– T cells and were euthanized. Upon necropsy, it was determined that TCR{alpha}-KO mice receiving IL-2-KO CD4+CD25– T cells were anemic (Fig. 2A) and had developed serum anti-erythrocyte Abs (Fig. 2B). In contrast, recipients receiving wild-type CD4+CD25– cells did not develop anemia nor anti-erythrocyte Abs within the 8-wk time frame of these experiments. Mice receiving wild-type CD4+CD25– T cells developed colitis within 3–4 mo, as published (15), but still did not develop AIHA. Recipients of IL-2-KO T cells also displayed elevated serum IgG2a levels, similar to IL-2-KO mice (Fig. 2C). Furthermore, transfer of IL-2-KO, but not wild-type, CD4+CD25– T cells led to lymphoproliferative disease in TCR{alpha}-KO recipients, with an accumulation of activated T cells (Fig. 2, D–F, and data not shown). B cells from the recipients of CD4+CD25– IL-2-KO T cells were also activated, as determined by CD5 and CD69 surface expression (data not shown). These data indicate that CD4+ T cells from IL-2-KO mice are sufficient to transfer disease in the absence of regulatory T cells and to stimulate autoantibody production by wild-type B cells. They also establish a central role for Th cells in this disease and suggest that normal B cells are not tolerant to erythrocyte Ags.


Figure 2
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  		FIGURE 2. Adoptive transfer of IL-2-deficient CD4+ cells is sufficient to transfer autoimmune hemolytic anemia. A total of 5 x 106 CD4+CD25– IL-2-KO (IL-2–/–) or wild-type (WT) cells was adoptively transferred into TCR{alpha}-KO (TCR{alpha}–/–) mice by tail vein injection. The mice were then followed for development of AIHA. At 4–6 wk postadoptive transfer, mice became moribund and were euthanized and compared with 19- to 25-day-old WT and IL-2–/– mice. A, Hematocrit of WT, IL-2–/–, TCR{alpha}–/– mice adoptively transferred with WT CD4+CD25– (WT into TCR{alpha}–/–) or IL-2–/– CD4+CD25– cells (IL-2–/– into TCR{alpha}–/–). WT vs WT into TCR{alpha}–/–, and IL-2–/– vs IL-2–/– into TCR{alpha}–/– hematocrit are not statistically different (indicated by *). Serum anti-erythrocyte Abs (B) and IgG2a (µg/ml; C) levels were evaluated by flow cytometry and ELISA, respectively. B, •, Represent serum from WT into TCR{alpha}–/–; {circ}, represent serum from IL-2–/– into TCR{alpha}–/– mice. D, Total lymph node cell numbers; E, CD4+ lymph node cell numbers; and F, percentage of CD4+ lymph node T cells expressing the surface activation marker CD44 were determined by flow cytometry. Representative data from one experiment of three performed. Each experiment used at least three mice per group.

 
Eliminating CD28 or CD40L delays progression of AIHA in IL-2-KO mice

To test whether blocking T cell activation or Th cell signals can delay or prevent disease, we crossed the IL-2-KO mice with mice lacking two molecules involved in the activation of lymphocytes, CD28 and CD40L. Kaplan-Meier survival curves of IL-2-KO, IL-2/CD28-KO, and IL-2/CD40L-KO mice showed that elimination of CD28 from IL-2-KO mice increased the median survival to 3.5 mo, whereas elimination of CD40L increased the median survival to greater than 6 mo (Fig. 3A and data not shown). Complete blood counts from these mice indicated that IL-2-KO mice developed anemia at less than 4 wk, whereas none of the IL-2/CD28-KO or IL-2/CD40L-KO mice developed anemia at any time point (Fig. 4B and data not shown). Elimination of CD28 or CD40L in IL-2-KO mice also markedly reduced serum IgG2a levels at 3–5 wk, and eliminated anti-erythrocyte Abs (Fig. 3C; data not shown). Thus, the CD28 T cell costimulatory pathway as well as the CD40L-dependent B cell activation pathway are necessary for development of this autoantibody-mediated disease.


Figure 3
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  		FIGURE 3. Eliminating CD28 or CD40L costimulation prevents autoimmune hemolytic anemia in IL-2-deficient mice. A, Kaplan-Meier survival plot of wild-type (WT), IL-2-KO, IL-2/CD28-KO, and IL-2/CD40L-KO mice. Survival studies were performed using at least six mice per genotype. B and C, Terminal bleeds of mice were performed at <5 wk and at 3 mo. Hematocrit (B) and µg/ml IgG2a (C) were evaluated at <5 wk. Three to six mice of each genotype were bled for testing.

 
In addition to developing AIHA, IL-2-KO mice also develop a lymphoproliferative disorder, which was marked by an increase in the accumulation of activated cells in the lymph node and spleen ((9) (Fig. 4). At 3–5 wk, elimination of CD28 from IL-2-KO cells resulted in a decrease in splenic and lymph node cell numbers, as well as a decrease in the percentage of CD44high and CD69high CD4+ T cells. However, by 3 mo of age, the IL-2/CD28-KO mice exhibited an increased number of lymph node and splenic cells with a rise in CD4+ T cell activation similar to that seen in young IL-2-KO mice. These results are consistent with the requirement for CD28 in T cell activation because there is a delay in the lymphoproliferative disorder and T cell activation in the absence of CD28 costimulatory signals. Tissues from IL-2/CD28-KO mice showed evidence of severe periductal inflammation in the pancreas and mild inflammation in the intestines, suggesting immune attack against multiple tissues. In fact, the IL-2/CD28-KO mice die at 3–5 mo. It is likely that death is secondary to the slow T cell expansion and tissue inflammation.

In contrast, IL-2/CD40L-KO T cells were activated and showed an increase in cell number as early as 3 wk, similar to the expansion of IL-2-KO T cells, with a large percentage of CD4+ cells expressing both CD69 and CD44 (Fig. 4). Therefore, although IL-2/CD40L-KO mice did not develop AIHA, they did have an expanded lymphocyte population. These data indicate, that whereas the T cells in the IL-2/CD40L-KO mice are activated and expanded, the B cells in these animals are not being stimulated to produce Abs. It is noteworthy that eliminating CD40:CD40L interactions abolishes autoantibody production even in the presence of massively expanded, presumably self-reactive, Th lymphocytes.

Because a major function of IL-2 is the maintenance of Tregs, we next evaluated the percentage of Foxp3+ CD4+ T cells in the spleen, lymph node, and thymus of IL-2-KO, IL-2/CD28-KO, and IL-2/CD40L-KO mice. As expected, we observed a 50% decrease in the percentage of CD4+Foxp3+ cells in the spleen and lymph nodes of IL-2-KO mice (Fig. 5). In the IL-2/CD28-KO mice, there were few CD4+Foxp3+ cells in the lymph node, spleen, or thymus. In contrast, the percentage of these cells in the IL-2/CD40L-KO mice was only slightly lower than in IL-2-KO mice. These data suggest that IL-2 and CD28 signals each contribute to the development and/or survival of Tregs. Thus, AIHA is less severe in the IL-2/CD28-KO mice than in IL-2-KO mice, even though the former contain even fewer Tregs. The likely explanation is that loss of CD28 from responding cells prevents their expansion and differentiation into pathogenic effector cells.


Figure 5
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  		FIGURE 5. Regulatory T cell percentages. Lymphocytes from 18- to 25-day-old mice were analyzed by flow cytometry. A, CD4+-gated cells expressing CD25 and Foxp3. B, Bar graphs indicate the mean percentage of CD4+ cells expressing Foxp3, and brackets show SDs of four Ab stains.

 
Blockade of IL-7R signals delays AIHA in IL-2-KO mice

One of the intriguing features of the IL-2-KO model is that massive T cell expansion and activation occur in the absence of IL-2, the prototypic T cell growth factor. The question thus arises: what, if any, growth factors compensate for the absence of IL-2 in driving these T cell responses? We postulated that IL-7, a growth factor known to promote the survival of naive and memory T cells, albeit not as potently as IL-2 (16, 17, 18) (reviewed in Ref. 19), is involved in the activation of autoreactive T cells in the IL-2-KO mice. The majority of IL-2-KO CD4+ T cells express higher levels of IL-7R{alpha} than naive wild-type CD4+ T cells, much like the expression pattern seen on memory CD4+ T cells (Fig. 6A). This finding suggests that IL-2-KO T cells may be responsive to IL-7. To test the role of IL-7 in the development of AIHA and lymphoproliferative disease, IL-2-KO mice and wild-type littermate controls were dosed with a mAb against IL-7R{alpha} or a control Ab beginning when the T cells are known to be in a resting state on day 7 (9). Strikingly, IL-2-KO mice treated with IL-7R-blocking Ab had an increased survival rate (Fig. 6B), lower titers of anti-erythrocyte Abs, and demonstrated diminished anemia compared with untreated IL-2-KO mice (Fig. 6, C and E). Serum IgG2a and IgG1 levels, however, remained elevated in treated IL-2-KO mice (Fig. 6D and data not shown). The decrease following treatment in anti-erythrocyte Abs, but not serum IgG or IgM levels, suggests that IL-7R blockade may preferentially eliminate or impede the autoreactive response.


Figure 6
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  		FIGURE 6. IL-7R{alpha}-blocking Ab delays onset of autoimmune hemolytic anemia and increases survival time of IL-2-deficient mice. Littermate mice were injected with 20 µg of IL-7R{alpha}-blocking Ab (A7R34) per gram weight of mouse i.p. three times per week. A, IL-7R surface expression on wild-type (open histogram) or IL-2-KO (filled histogram) CD4+ T cells in the lymph node without Ab treatment. B, Kaplan-Meier survival curve. Survival was monitored until mice were moribund. C–E, Mice were euthanized at 21 days, and hematocrit (C), serum IgG2a (D), and anti-erythrocyte Ab levels (E) were determined. C and D, Bars represent the mean. E, {circ}, Represent serum from IL-2-KO mice without treatment; •, represent serum from IL-2-KO mice treated with IL-7R{alpha} Ab. An unpaired Student’s t test was used to determine statistical significance (*, indicates p = 0.018). Data from at least six mice per group collected during four experiments.

 
IL-7R Ab-treated mice also showed a decrease in lymph node cell numbers (Fig. 7A), indicating a suppression or delay of lymphoproliferation. This decrease in total lymph node cell numbers was due to a decrease in both CD4+ T cells (data not shown) and B cells (Fig. 7C). Importantly, the percentage of activated CD4+ T cells in Ab-treated IL-2-KO mice dropped by ~50% to near wild-type levels (Fig. 7B). Although the total CD4+CD44high cell numbers remained elevated in IL-7R-treated IL-2-KO mice (3.8 x 106 cells) compared with untreated wild-type mice (1.0 x 106 cells), this was a large decrease relative to the number of activated cells in untreated IL-2-KO mice (14.2 x 106 cells). The percentage of CD8+CD44high cells did not change with Ab treatment, indicating that the IL-7R Ab did not eliminate T cells by Ab-dependent cell-mediated cytotoxicity. This result is consistent with other published studies using this dose of Ab (20).

Because CD4+ T cells are diminished and display a less activated phenotype with treatment, loss of these cells most likely plays a role in the delay of AIHA. This delay may be due, in part, to the drop in B cell numbers and the decrease in anti-erythrocyte Ab levels. However, serum IgG2a levels in the treated mice are still elevated, and normal IgM levels are maintained (Fig. 6D and data not shown), indicating that Ab-producing cells persist in these mice. Thus, IL-7R Ab treatment may predominantly block the erythrocyte Ag-specific response. It is also possible that autoantibodies bind to self Ags and are cleared more rapidly than other Igs, and for this reason treatments that reduce Ab production have a greater apparent effect on Ag-specific Ab levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Autoimmune hemolytic anemia is a disease caused by autoantibodies against several Ags expressed on erythrocytes. AIHA can occur alone, but is more often seen in association with other autoimmune diseases, cancer, drug treatment regimens, transfusion, and pregnancy. Although the incidence is relatively low at 1 in 80,000 (~3,400 people in the U.S. every year), the mortality rate remains high at 11.2%, most likely due to inadequate knowledge of the immunology of AIHA (21). Thus, it is a serious systemic autoimmune disease for which there are no workable biological therapies other than splenectomy and general immunosuppressive and anti-inflammatory drugs.

The IL-2-KO mouse provides a powerful model for defining the signals involved in the development of spontaneous autoimmune disease in the absence of regulatory T cells. AIHA in these mice is mediated by Th cell-dependent Abs, and based on our data presented in this work, we believe it is amenable to rational therapeutic manipulations. Although there is no analogous human example of complete IL-2 deficiency, there are many human diseases, including systemic lupus erythematosus, multiple sclerosis, Crohn’s, and diabetes (22, 23), in which autoimmunity is attributed to decreased regulatory T cell numbers or suppressive function.

TCR{alpha}-KO mice receiving CD4+CD25– IL-2-KO T cells develop AIHA and lymphoproliferative disease within 4–8 wk, suggesting that CD4+ T cells play a pivotal role in disease progression. Previous work by others has shown that both B and T cells are critical for AIHA development, as Rag-KO mice are protected from disease. Although B cells are necessary for autoantibody production in this antibody-mediated disease, it is important to note that adoptive transfer of CD4+CD25– IL-2-KO T cells into TCR{alpha}-KO mice stimulated wild-type B cells to produce erythrocyte autoantibodies, indicating that only self-reactive T cells are required for AIHA initiation. This necessary role for Th cells suggests that targeting T cell activity may benefit the treatment of this autoimmune disease. However, this finding does not rule out the possibility that B cells may influence disease progression by diversifying the T cell repertoire and enhancing epitope spreading, as has been previously described in diabetes (24).

This study demonstrates that in the absence of regulatory T cells, costimulatory and common {gamma}-chain cytokine signals are required for the spontaneous activation and survival of self-reactive T lymphocytes in IL-2-KO mice. Erythrocyte-directed autoreactivity is abolished by the elimination of B or T cell activation, as indicated by the removal of CD28 or CD40L function. The differences in disease manifestation observed between IL-2/CD28-KO and IL-2/CD40L-KO mice establish that lymphoproliferation and autoimmunity can be segregated. It is striking that autoimmune disease does develop in the absence of CD28, although it is diminished and is not erythrocyte directed, but that autoimmunity fails to develop in the absence of CD40L. This result suggests that blocking the CD40:CD40L pathway may be a more effective strategy for treating autoantibody-mediated diseases than blocking B7:CD28 signals.

In addition to its normal role in the survival of naive and memory T cells, IL-7 signaling also appears to contribute to the activation of self-reactive T cells in the absence of IL-2. This role for IL-7 is somewhat surprising, because IL-7 is not thought to be an essential activator of autoreactive T cells. Our observations may have a number of explanations, as follows: 1) that IL-7 signaling compensates for the loss of IL-2 in T cell expansion or survival; 2) that IL-7’s significance becomes apparent only when IL-2 or regulatory T cells are absent or ineffective; or 3) that effector/memory-like CD4+CD44high T cells accumulate in IL-2-KO mice because of aberrant IL-7R expression, resulting in a survival advantage. These results raise the possibility that blocking IL-7 signaling may provide therapeutic benefit for patients suffering from AIHA and, perhaps, other autoantibody-mediated diseases.

IL-7R Ab treatment significantly delayed disease onset and development of spontaneous AIHA. Although there was only partial protection, blocking IL-7R signaling during development of AIHA and lymphoproliferative disease produced a significant ameliorative effect in the IL-2-KO mice. There are a number of possibilities as to why blockade of IL-7R signaling was not sufficient for complete disease protection. First, it is possible that blockade of IL-7R signaling is incomplete, even though following IL-7R Ab treatment, very few lymphocytes continued to express surface IL-7R (data not shown). A second possibility is that over time T cell clones emerge that are less dependent on IL-7R signaling. In addition, the delay in disease due to IL-7R blockade might suggest that autoreactive cells newly activated in the periphery play a critical role in autoimmunity. These naive CD4+ T cells would express high levels of IL-7R and might be preferentially prevented from becoming activated and expanded during the Ab treatments. It remains to be seen whether blocking IL-7 signals will be a feasible therapeutic strategy, given the role of this cytokine in the maintenance of normal naive and memory T cells. It may be that antagonists can be titrated to inhibit autoreactive lymphocytes without affecting normal cells. Whether IL-7 antagonism will be effective in the presence of IL-2 is also an open question.

Taken together, our results demonstrate profound inhibition of Ag-specific T and B cell responses by elimination of costimulation and IL-7R signaling, as observed by elimination of erythrocyte Abs and delay of AIHA. In addition, we demonstrate that autoreactive cells use the same signals that conventional cells require for activation and survival, namely CD28 for activation, CD40L for B cell help, and the common {gamma}-chain cytokine, IL-7, for survival. Elimination of any of these pathways delays the onset of autoimmune disease presumably by reducing the survival or activation state of the self-reactive lymphocytes. These results suggest that elimination of costimulatory or common {gamma}-chain signals may be effective in the treatment of AIHA and warrants further evaluation.


   Acknowledgments
 
We thank members of the Abbas laboratory for helpful discussions, Dr. S. Smith for critical comments on the manuscript, P. Lin for technical assistance, S. Jiang for cell sorting, and C. Benitez for mouse typing.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
Current employee (part- or full-time) or contractor


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants P01AI35297 and T32A107334-17. Back

2 Address correspondence and reprint requests to Dr. Abul K. Abbas, Department of Pathology, University of California M-590, 505 Parnassus Avenue, San Francisco, CA 94143. E-mail address: abul.abbas{at}ucsf.edu Back

3 Abbreviations used in this paper: AIHA, autoimmune hemolytic anemia; KO, knockout; Tregs, regulatory T lymphocytes. Back

Received for publication February 13, 2007. Accepted for publication June 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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