of June 29, 2015.This information is current as

Hemopoiesis, and Apoptosisamong Regulatory T Cells, Inflammation,

Knockout Mice: InterplayαBiology of IL-2RThe Role of Fas in the Immune System

J. Sung and Shyr-Te JuRahul Sharma, Harini Bagavant, Wael N. Jarjour, Sun-Sang

http://www.jimmunol.org/content/175/3/1965doi: 10.4049/jimmunol.175.3.1965

2005; 175:1965-1973; ;J Immunol 

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, 17 of which you can access for free at: cites 31 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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The Role of Fas in the Immune System Biology of IL-2R�Knockout Mice: Interplay among Regulatory T Cells,Inflammation, Hemopoiesis, and Apoptosis

Rahul Sharma, Harini Bagavant, Wael N. Jarjour, Sun-Sang J. Sung, and Shyr-Te Ju1

Introducing lpr mutation prevents early mortality associated with IL-2R� knockout (KO) mice, prompting us to determine therole of Fas in the immune system biology of IL-2R� KO mice. Consistent with a defect in CD4�CD25� regulatory T (Treg) cellexpression, spontaneous lymphocyte activation in lymphoid organs was observed in 6-wk-old mice. In 16- to 22-wk-old mice,infiltration of leukocytes was observed in bone marrow, colon, lung, pancreas, lacrimal gland, and salivary gland, but not in heart,thyroid, liver, stomach, small intestine, ovary, and kidney. In the lymphocytes-infiltrated bone marrow, B cell lymphopoiesis wasblocked at pro-B to pre-B/immature B stage, culminating in an age-dependent B cell loss in the periphery. These phenotypes werealso observed in IL-2R� KO mice bearing the lpr mutation (DM mice), indicating Treg cell function and the phenotypes attributeddirectly to Treg cell abnormality are largely Fas-independent. However, anemia and body weight loss were partially prevented,tissue cell apoptosis was inhibited, and lifespan was improved in the DM mice, demonstrating Fas-dependent elements in theseprocesses. Our age-dependent, lifelong analysis of IL-2R� KO and DM mice supports a CD4�CD25� Treg cell-based mechanismfor the abnormal immune system biology observed in IL-2R� KO mice and provides a global view of the interplays among Tregcells, multiorgan inflammation, hemopoiesis, and apoptosis. The Journal of Immunology, 2005, 175: 1965–1973.

T he IL-2/IL-2R signaling pathway plays a critical role inthe immune system (reviewed in Refs. 1–3). It is a pow-erful signal for lymphocyte proliferation and a critical

factor for the development and expansion of the CD4�CD25�

regulatory T (Treg)2 cells that down-regulate T cell proliferation/activation. A characteristic phenotype of Treg cell absence is tissueinflammation (4–7). Targeted mutation of IL-2, IL-2R� (CD25),or IL-2R� (CD122) gene results in a similar phenotype character-ized not only by tissue inflammation as exemplified by ulcerativecolitis but also by severe anemia, body weight loss, and otherimmune abnormalities such as B cell deletion reported in old IL-2knockout (KO) mice and IL-2R� KO mice (8–15). A consequenceassociated with these abnormalities is early mortality, which pre-vents a thorough analysis of the immune system defect and im-mune regulation in these mice, especially in adult and older mice.

The FasL (CD178)/Fas (CD95)-mediated apoptosis pathway iscritical to the maintenance of peripheral tolerance, most notably insystemic autoimmune disease such as the lupus-like autoimmunedisease in lpr and gld mice (16, 17). In addition, FasL on infil-trating lymphocytes of the inflamed tissues have been shown toinduce apoptosis of tissue cells (18–21). We recently have shownthat introducing the lpr mutation into IL-2 KO mice prolongs theirlifespan (21). This is attributed at least in part to the prevention ofanemia and in part to the prevention of the FasL-induced apoptosisof colon epithelial cells. However, whether Fas-mediated signalingpathways are involved in a specific phenotype such as B cell de-

letion, tissue inflammation, tissue cell death (other than colon) wasnot determined (21).

It is likely that a common mechanism is responsible for many ofthe phenotypes shared among IL-2 KO, IL-2R� KO, and IL-2R�KO mice. We propose a hypothesis based on a current concept ofTreg cell function to explain the shared phenotypes among thesemouse strains. We hypothesize that the absence of Treg cells al-lows CD4�CD25� T cells (including autoimmune T cells) to bepolyclonally activated. Activated autoimmune T cells infiltratespecific target organs where they interfere with organ functions,leading to various phenotypes. In addition, autoimmune T cellsthat target bone marrow are induced, and bone marrow is highlysensitive to these cells. T cell infiltration into bone marrow sup-presses bone marrow function, leading to progressive hemopoie-sis-based abnormalities. We choose to study IL-2R� KO mice be-cause T cell infiltration into bone marrow, B cell deletion, andinflammation in multiple organs were not described (13). Also, weintroduced lpr mutation into IL-2R� KO mice (referred to as DMmice) to determine whether a specific phenotype is dependent onTreg cells or Fas expression. This is significant because Fas mu-tation prolonged the lifespan of IL-2R� KO mice. Fas mutation didnot prevent inflammation in multiple organs. However, it affectedthe pathology of the inflamed lung and colon. Like IL-2 KO andIL-2R� KO mice, we also observed T cell infiltration into bonemarrow where erythropoiesis and B cell lymphopoiesis were im-paired, culminating a progressive, age-dependent anemia and pe-ripheral B cell deletion. Collectively, our study provides a mech-anism-based description of the immune system biology throughoutthe lifespan of IL-2R� KO mice by characterizing their specificimmune phenotypes and by defining the roles of CD4�CD25�

Treg cells and Fas in these phenotypes.

Materials and MethodsBreeding and genotyping

C57BL/6.Il2R��/� mice and B6.MRL-Faslpr/J (B6.lpr or lpr�/�) micewere obtained from The Jackson Laboratory. Because Il2R��/� (IL-2R�KO) mice are sterile, Il2R��/� mice were first bred with lpr�/� mice to

*Division of Rheumatology and Immunology, Department of Internal Medicine, Uni-versity of Virginia, Charlottesville, VA 22908

Received for publication February 28, 2005. Accepted for publication May 5, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Division of Rheu-matology and Immunology, Department of Internal Medicine, University of Virginia,Charlottesville, VA 22908-0412. E-mail address: sj8r@virginia.edu2 Abbreviations used in this paper: Treg, regulatory T; KO, knockout.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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obtain Il2R��/�lpr�/� F1 offspring. F1 mice were intercrossed to obtainIl2R��/�lpr�/� and Il2R��/�lpr�/� mice. We then intercrossed Il2R��/

�lpr�/� mice to increase the frequency of Il2R��/�lpr�/� offspring. TailDNA preparations of 4-wk-old mice were used for genotyping by PCR.The primers and condition used for fas PCR analysis have been described(21). The Il2R� gene was analyzed by PCR using the primer sequences andreaction conditions from The Jackson Laboratory website (�www.jax.org�).PCR products were determined by their sizes as follows: 218 bp for fasmutant of lpr mice, 182 bp for the normal fas, 280 bp for the Il2R� mutantallele, and 146 bp for the normal Il2R� allele. DNA samples fromC57BL/6J (B6), IL-2R� KO, and B6.lpr mice were used as controls. Micewere housed at the University of Virginia animal facility, and experimentswere conducted following the protocol approved by the Institutional Ani-mal Care and Use Committee.

General examination of mice

Mice were examined twice weekly for mortality. Starting at 4 wk of age,mice were weighed and blood samples were collected every 2 wk. Tenmicroliters of blood samples were collected and immediately diluted in 90�l of cold PBS, pH 7.2. Aliquots were used to determine hemoglobin levelsand cell-free samples were collected by centrifugation and used for thedetermination of IgG levels using the mouse IgG ELISA kit (BethylLaboratories).

Determination of hemoglobin level

Blood samples were diluted 100-fold with dilution buffer (3 mM potassiumferricyanide, 1.5 mM potassium cyanide, 5 mM sodium borate, 0.1% Non-idet P-40) in which RBC were lysed and the released hemoglobin wasconverted to cyanomethemoglobin. The relative hemoglobin concentra-tions were determined photometrically at 546 nm.

Immunohistochemical and immunofluorescence staining

Sections of paraffin-embedded tissues were stained with H&E. Tissues ex-amined are lungs, thyroids, lacrimal glands, salivary glands, hearts, stom-achs, livers, pancreas, kidneys, small intestines, colons, and ovaries. Ap-optosis was determined using a TUNEL assay kit (Apoptag PlusPeroxidase In Situ Apoptosis Detection kit; Serologicals). We followed themanufacturer’s protocol and included the positive control provided by themanufacturer. Background staining was determined by incubation withbuffer in the absence of TdT. Slides were counterstained with methyleneblue followed by graded alcohol dehydration and mounting. TUNEL� cellsdeveloped a characteristic brown/black color in the nuclear region.

Confocal microscopy

Frozen sections of lymph nodes were stained for 30 min with PE-conju-gated anti-B220 (RA3-6B2) and hamster anti-CD3 (145-2C11) (BD Bio-sciences). After washing, slides were incubated for 30 min with Alexa647-conjugated goat anti-hamster Ab (Molecular Probes). For a negativecontrol, we used rat IgG and hamster IgG (both from BD Biosciences) inplace of anti-B220 mAb and anti-CD3 mAb, respectively. The slides wereexamined using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss).

Flow cytometric analysis

Lymphocytes in blood, lymph nodes, spleen, bone marrow, and thymuswere analyzed. Single cell suspensions of samples were treated with am-monium chloride to remove erythrocytes, washed, and then stained forvarious cell surface markers. Cells (106) were suspended in 100 �l of PBScontaining 4% BSA and incubated with 1 �g of various fluorescent Abs for30 min at 4°C. FITC- and/or PE- conjugated anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-Thy-1.2 (30H12), anti-B220 (RA3-6B2), anti-CD43 (S7), anti-Fas (Jo-2), and anti-CD69 (H1.2F3) mAb wereobtained from BD Biosciences. FITC conjugated goat anti-mouse IgM waspurchased from Southern Biotechnology Associates. At least 104 stainedcells were analyzed using a FACScan (BD Biosciences) equipped withCellQuest (BD Biosciences). Post acquisition analyses were conducted us-ing FlowJo software (Tree Star).

ResultsFas mutation prevents early mortality and prolongs the lifespanof IL-2R� KO mice

We introduced the fas mutant gene of B6.lpr mice into IL-2R� KOmice by breeding. The genotypes of progeny were identified byPCR on tail DNA samples (Fig. 1A). The cell surface phenotypeswere demonstrated by staining lymphocytes with FITC-anti-Fas

mAb for Fas expression and FITC-anti-CD4 plus PE-anti-CD25mAb for Treg cells (Fig. 1, B and C). Mice were observed weeklyfor general appearance and mortality. In addition, body weightgain/loss and the severity of anemia were determined. The com-piled data are shown in Fig. 2. Mice bearing both Fas and IL-2R�-targeted mutations (DM mice) survived significantly longer thanIL-2R� KO mice but did not reach to the life span of B6.lpr andB6 mice (Fig. 2A). Severe anemia as defined by �50% of controlB6 hemoglobin level was observed in 10-wk- but not 4-wk-oldIL-2R� KO and DM mice (Fig. 2B). Interestingly, the protectionof anemia by lpr mutation in DM mice is moderate for males andunremarkable for females (Fig. 2B). The reason for the differentialprotection is unclear. A moderate protection of body weight loss ascompared with IL-2R� KO mice was observed in male DM micebut not in the female DM mice (Fig. 2C). Still, Fas mutation pro-longed the lifespan of female mice as well as male mice. Thepartial and marginal improvement of body weight gain and theweak prevention of anemia agree with the inability of these miceto reach the lifespan of B6.lpr and B6 mice.

Spontaneous T cell activation in 6-wk-old IL-2R� KOand DM mice

We determined whether spontaneous T cell activation occurs as aresult of lifelong absence of the CD4�CD25� Treg cells. Asshown in Fig. 3, when compared with age- and sex-matched B6mice, a significantly higher proportion of the CD4� T cells in the

FIGURE 1. Genotyping and phenotyping IL-2R� KO and DM mice. A,The PCR product sizes are 218 and 182 bp for the lpr and wild-type fasallele, respectively. The PCR product sizes for the mutant and wild-typeIL-2R� are 280 and 146 bp, respectively. In this example, lane 1 and lane8 represent DM mice, lane 3 has the IL-2R� KO genotype, lane 5 is lpr,and lane 7 is a wild-type mouse. Lane 6 is molecular size standard. B,Lymph node cells of genotyped mice were stained with FITC-anti-FasmAb for Fas expression and analyzed with flow cytometry (shaded, iso-type; thin line, DM; thick line, B6). C, The genotyped DM mouse is neg-ative for CD4�CD25� Treg cells.

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lymph nodes of 6-wk-old IL-2R� KO (6 of 12 or 50%) and DM (7of 18 or 39%) mice expressed the activation marker CD69,whereas in B6 mice the value was 16%. Similarly, increase inCD69 expression was observed for CD8� T cells. We noted thatthere was an increase in CD8� T cells in both IL-2R� KO and DMmice (data not shown) and the proportion of CD69� cells withinthe CD8� T cell population was somewhat less than the CD69�

cells in the CD4� T cell population.

Fas mutation did not prevent tissue inflammation

We determined whether IL-2R� KO mice (3–4 mo old) developinflammatory response in multiple tissues/organs following thespontaneous T cell activation. Various tissues were collected fromB6, B6.lpr, IL-2R� KO, and DM mice and the stained tissue slideswere examined. As shown in Fig. 4, IL-2R� KO and DM miceshowed histopathological changes in lungs, colon, pancreas, andlacrimal glands in comparison to the counterparts of B6 and B6.lpr(data not shown) mice. Both IL-2R� KO and DM mice developedlung inflammation. The severity of inflammation was greater in theIL-2R� KO mice compared with DM mice. Both showed cellularinfiltrates surrounding the bronchiolar lumen extending into thelung parenchyma. However, there were differences in the nature ofthe infiltrating cells. Lymphocytes were the major infiltrating cellsin all six DM mice examined. In three of the six IL-2R� KO miceexamined, lymphocytes were also the major cell type in the af-fected regions. However, strong infiltration of both neutrophils andlymphocytes were observed in the other three mice. In some areas,the neutrophil-containing exudates formed a plug in the bronchio-

lar lumen along with hypertrophy of the lining epithelium. Thereason why neutrophil infiltration was observed in the lung ofsome but not all IL-2R� KO mice is not clear at present but itspresence is consistent with the idea that FasL is a potent chemo-tactic factor for neutrophils (22).

The colonic mucosa in both IL-2R� KO and DM mice showedsignificant thickening and hypertrophy compared with control B6mice (Fig. 4). Lymphocytic infiltration in the lamina propria was ob-served in both groups but was more prominent in the DM mice.Multinucleated giant cells were also observed in the colonic mucosaof IL-2R� KO mice. The submucosa and muscular layers appearednormal.

Changes in the pancreas and lacrimal glands were characterizedby extensive lymphocytic infiltration in the periductal regions.There was little involvement of the exocrine glands. In the pan-creas, infiltration did not appear to invade the pancreatic islets orcause islet cell destruction. These mice did not develop diabetesmellitus as determined by urinary glucose level (data not shown).Similar periductal infiltration was seen in salivary glands of bothIL-2R� KO and DM mice. Inflammation in thyroid, liver, stom-ach, small intestine, ovary, kidney, and heart was either very mildor not observed (data not shown). No pathological changes wereapparent in tissues from age-matched control B6 and B6.lpr mice.These data suggest that inflammation in multiple tissues/organs isdue to the lack of CD4�CD25� Treg cells in both IL-2R� KO andDM mice. In addition, Fas deficiency in DM mice influences thequalitative nature (infiltrating cell type) and extent of inflammation(colon in DM mice) of the inflammatory response.

Fas mutation inhibited apoptosis of tissue cells in the inflamedlung and colon

To determine whether tissue cells in the severely inflamed siteswere killed by Fas/FasL-mediated apoptosis, we used the TUNELassay to determine the cell types and the number of apoptotic cellsin the lung and colon of IL-2R� KO and DM mice. A represen-tative experiment is shown in Fig. 5. Few apoptotic cells wereobserved in the lung of a B6 mouse. Many apoptotic cells wereobserved in the lung of the 16-wk-old IL-2R� KO mouse. Al-though many of them were leukocytes, apoptotic tissue cells wereevident (inset and right panels). In the lung of an age-matched DMmouse, the number of apoptotic leukocytes was significantly re-duced in comparison with IL-2R� KO mouse. In addition, apo-ptotic tissue cells were rarely observed (inset and right panels).

FIGURE 2. Fas mutation protects IL-2R� KO mice from early mortality,anemia, and body weight loss. The DM mice lived longer than IL-2R� KOmice (A) and had improved blood hemoglobin levels (B, male; C, female) andbody weight (D, male; E, female). The number of mice examined are equal toor more than six (n � 6) except for IL-2R� KO mice (n � 5 for male and n �3 for female).

FIGURE 3. Spontaneous activation of T cells in IL-2R� KO and DMmice. Lymph node cells from 6-wk-old female B6, IL-2R� KO, and DM micewere stained with anti-CD4 mAb plus anti-CD69 mAb (upper panels) andanti-CD8 mAb plus anti-CD69 mAb (lower panels). Please note that the num-bers in the quadrants are percentages of the population of total lymphocytes.

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Similarly, a higher number of apoptotic cells were detected in theinflamed colon of IL-2R� KO mice (Fig. 5). Both apoptotic cellsand tissue damage were less evident in the inflamed colon of DMmouse than IL-2R� KO mouse. These observations suggest thatFasL is induced in IL-2R� KO mice and that Fas-mediated apo-ptosis plays a major role in cell death during the inflammatoryresponse in IL-2R� KO mice.

IL-2R� KO mice display Fas-independent, age-dependentdeletion of B cells

Blockade of B cell differentiation in bone marrow. Previousstudies of IL-2 KO and IL-2R� KO mice have shown that B celllymphopoiesis was severely blocked at the pro-B to pre-B/imma-ture B cell stage as a result of infiltration of lymphocytes andleukocytes, respectively (12, 14). We also observed a strong infil-tration of T cells in the bone marrow of IL-2R� KO and DM mice(see Table III). Therefore, we examined B cell lymphopoiesis inthe bone marrow of both the IL-2R� KO (16 wk old) and DM (22wk old) mice using anti-B220, anti-IgM, and anti-CD43 mAb todetermine the B cell subpopulations at various differentiationstages in the bone marrow. A representative experiment is shown

in Fig. 6. The percentages of B220�IgM� pre-B/immature B cells(from 6.13% in B6 to 0.36% in IL-2R� KO and 1.03% in DMmice) and B220�IgM� mature B cells (from 3.01% in B6 to0.33% in IL-2R� KO and 0.58% in DM mice) were greatly re-duced in the bone marrow of IL-2R� KO and DM mice, whereasthe percentage of B220�CD43� pro-B/small pre-B cells was notsignificantly different from a B6 control (4.47% in B6 vs 3.69% inIL-2R� KO and 4.75% in DM mice). The data indicate that bonemarrow B cell differentiation is blocked at the pro-B to pre-B/immature B cells stage and this phenotype is mostly Fas-indepen-dent. It is important to note that the total number of bone marrowcell count (after depletion of erythrocytes) was comparable amongthese mice (data not shown), indicating that the output of naive Bcells into the periphery is greatly reduced in the old IL-2R� KOand DM mice as a consequence of this blockade.

Depletion of B cell zone in lymph nodes. We reasoned that adirect consequence of lacking an input of B cells from bone mar-row could result in B cell depletion in the periphery. Following theimmature B cell differentiation blockade in the bone marrow, thearea of B cell zone in the lymph nodes of 14-wk-old mice was

FIGURE 4. Histological examina-tion of tissues from B6, IL-2R� KO,and DM mice. Various tissues werefixed in 10% neutral buffered forma-lin, paraffin embedded, and sectioned,and 4-�m sections were stained withH&E. Lung (A–C), colon (D–F), pan-creas (G–I), lacrimal glands (J–L),and thyroid (M–O) are shown. Lym-phocytic infiltration (ly) seen in IL-2R� KO and DM mice is indicated.IL-2R� KO mice have a strong neu-trophil infiltration (arrows) in thelungs and bronchiolar lumen exudate(B). Arrow in inset in E shows amultinucleated giant cell seen in thecolonic mucosa of IL-2R� KO mice.Photomicrographs were taken at�200 for lung (A–C) and colon (D–F). All other photomicrographs weretaken at �100 magnification. Little orno infiltration was observed in tissuesof control B6 mouse.

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proportionally reduced as demonstrated by two-color staining us-ing anti-B220 mAb/anti-CD3 mAb and examined with a confocalmicroscope (Fig. 7). This reduction is significant considering thefact that the lymph node size of IL-2R� KO and DM mice wasmuch larger than B6 control. In addition, a more severe B cell zonedepletion was observed in 22-wk-old DM mice (data not shown).Interestingly, the remaining B cell zone was infiltrated with Tcells. Infiltration of T cells into B cell zone and loss of T/B celldemarcation was also observed in the B6.lpr lymph node as pre-viously reported (23). Consequently, both Treg cell abnormalityand Fas mutation could contribute to the T cell infiltration into Bcell zone in DM mice.

Depleting serum IgG caused by early B cell activation in IL-2R�KO mice and late B cell activation in lpr mice. Early polyclonallymphocyte activation and the subsequent B cell deletion should affectserum IgG levels. Therefore, the total serum IgG levels of mice atvarious ages were determined (Fig. 8). The IgG level of B6 mice didnot fluctuate much during the entire period of study. As early as 1 moold, the IgG level of IL-2R� KO mice was already significantlyhigher than B6. This level was maintained for a month and then de-clined for the next 2 mo to a level slightly lower than B6 control. TheIgG level of B6.lpr was normal up to 2 mo of age and sharply in-creased to as high as 350% of the IgG level of B6 mice. The age-dependent pattern of IgG expression level in the DM mice resemblesthat of the IL-2R� KO mice but not the B6.lpr mice (Fig. 8). Espe-cially, the increase in IgG level in old B6.lpr mice was not observedin the old DM mice. The serum IgG level in the 22-wk-old DM micedropped to 30% of the level of B6 mice at 9 mo of age (Fig. 8). Thedata demonstrated a dominant role of Treg cell abnormality over Fasmutation in regulating serum IgG level. This dominance is consistentwith the early lymphocyte activation and the late B cell deletion in theIL-2R� KO and DM mice.

The age-dependent elevation of IgG followed by the dramatic de-cline in the sera of IL-2R� KO and DM mice is consistent with thehypothesis that absence of Treg cells in these mice permitted an earlyT cell-dependent polyclonal activation of B cells, and a subsequentdeletion of B cells resulted from T cell infiltration into bone marrow.In addition, the deranged lymphoid architecture in these mice may not

FIGURE 6. B cell development arrest in the bone marrow of IL-2R�KO mice is Fas-independent. Bone marrow cells from B6, IL-2R� KO, andDM mice were stained with PE-anti-B220 mAb and FITC-anti-CD43 mAb.B cell development in bone marrow was affected at pre-B/immature B cellstage. Both B220highIgM� (mature B cells) and B220intIgM� (immature Bcells) cells were reduced in IL-2R� KO and DM mice (top row). TheB220�CD43� cells (late pre-B, immature B and mature B cells) wereaffected in both IL-2R� KO and DM mice, whereas the B220�CD43�

(pro-B and early pre-B) population was not (bottom row). The B6 and DMmice were 22 wk old while the IL-2R� KO mouse was 16 wk old. Arepresentative of three experiments is shown.

FIGURE 5. Fas mutation protects cell death in the inflamed lung and colon of IL-2R� KO mice. Sections of lung (top panels) and colon (bottom panels)were stained for apoptotic cells by TUNEL assay. TUNEL� cells as indicated by dark brown color were more frequent in the IL-2R� KO mice than DMmice (�100). The inset captured at �400 magnification shows the apoptotic cell types. Both apoptotic leukocytes (arrows) and apoptotic tissue cells(arrowheads) were observed. (Ly, lymphocyte; N, neutrophil). The bar graphs (right panels) show the numbers of TUNEL� tissue cells, TUNEL�

lymphocytes, and total apoptotic cells in the inflamed tissues. A total of five randomly selected fields were counted for each sample at a magnification of�200. The tissue apoptotic cells were distinguished from apoptotic lymphocytes on the basis of the size of the nucleus and surrounding cytoplasm.

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allow optimal T/B interaction and this may also contribute to theage-dependent decline in serum IgG level. Finally, the slower rate ofdecline of the serum IgG level in the DM mice in comparison toIL-2R� KO mice suggests a small role of Fas mutation in this phe-notype by protecting lymphocytes from FasL-mediated apoptosis.

Changes in peripheral B cell numbers.

Because we also observed changes in B cell proportion in the bloodsamples of IL-2R� KO and DM mice, the age dependence of thisphenotype was determined for individual mice by staining blood sam-ples obtained at various age points with anti-B220 and anti-Thy-1mAb (Fig. 9). At 4 wk of age, the earliest age analyzed, the percentage

of B cells in the lymphocyte population in blood was moderatelyreduced (70% of B6 control). This value rapidly declined to a plateauof 26% in 10-wk-old mice and 28% in 22-wk-old mice.

To determine the actual changes in B cell number and propor-tion in lymph nodes, spleen, and bone marrow, individual micewere euthanized at specific ages and single cell suspensions wereprepared and counted as described in Materials and Methods. Cellswere stained with PE-anti-B220 and FITC-anti-Thy-1 mAb, and

FIGURE 8. Age-dependent changes in serum IgG levels. Serum sam-ples from individual mice (n � 3) obtained at specific age were pooled. IgGlevels were determined by ELISA as described in Materials and Methods.The pattern of IgG levels of DM mice resembles that of IL-2R� KO micebut not B6.lpr mice. The IL-2R� KO and DM mice had very high serumIgG levels at a young age that progressively declined with age. Early mor-tality prevented sample procurement from IL-2R� KO mice �4 mo old.The data is a representative of three similar experiments.

FIGURE 9. IL-2R� KO and DM mice display a progressive, age-de-pendent reduction of B cells in the blood. Blood samples of individual B6,IL-2R� KO, and DM mice were obtained at various age points and stainedwith PE-anti-B220 and FITC-anti-Thy-1.2. IL-2R� KO mice did not sur-vive for the 22-wk sampling.

FIGURE 7. IL-2R� KO and DM mice display disrupted lymph node structure. Frozen sections of inguinal lymph nodes from B6, B6.lpr, IL-2R� KOand DM mice were stained for B cells (red) and T cells (blue) using PE-anti-B220 mAb and anti-CD3 mAb followed by Alexa 647-conjugated goatanti-hamster Ab, respectively (top panels) and examined under a confocal microscope. All mice used in this experiment were 14 wk old. Note the lymphnode size difference between B6 and mutant mice. The bottom panels focus on the B cell zones demarcated in the top panels. Reduction of B cell zonesand infiltration of T cells into the B cell zones were observed for B6.lpr, IL-2R� KO, and DM mice.

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the proportion was used to determine the total number of T and Bcells in each preparation. As shown in Table I, the relative per-centages of B cell population in lymph nodes of 4-wk-, 10-wk-,and 22-wk-old DM mice were reduced, respectively, to 82% (24 of28, p � 0.05), 53% (19 of 36, p � 0.05), and 7% (4 of 58, p �0.05) of age-matched control mice. The absolute number of B cellsin the 4-wk- (5 � 106 in DM vs 3 � 106 in B6) and 10-wk-old (4 �106 in DM vs 3 � 106 in B6) DM mice was not significantlydifferent from age-matched B6 control. A significant decrease inthe absolute B cell number was observed only in 22-wk-old mice(from 4 � 106 of B6 mouse to 1 � 106 of DM mouse), and thisagreed well with the reduction of serum IgG level in these mice.This pattern of B cell regulation is also observed in the spleen andin IL-2R� KO mice (4-, 10-, and 14-wk-old mice were analyzed

due to early mortality), indicating that B cell deletion in the pe-riphery is mostly Fas-independent. We also observed a progressive,age-dependent increase in Thy-1� T cells in IL-2R� KO and DMmice as compared with B6 control (Table I). This increase in Thy-1�

T cells is intrinsic to IL-2R� KO and is caused by a specific increasein CD8� T cells in IL-2R� KO and DM mice (data not shown).

Change in CD4� T cell numbers in bone marrow

CD4� T cells from IL-2 KO and IL-2R� KO mice have beenshown to transfer into nude mice several major phenotypes (11, 12,14). Therefore, we determined whether the absolute number ofCD4� T cells was increased in these mice, we used FITC-anti-CD4� mAb to stain CD4� T cells in the lymph nodes, spleen, andbone marrow of various mouse strains (Table II). For all lymphoidtissues studied except bone marrow and 22-wk-old DM mice, thetotal number of CD4� T cells was comparable to that observed inthe age-matched B6 controls, despite the fact that these CD4� Tcells expressed activated phenotype based on CD69 expression(Fig. 3). Although some activated CD4� T cells must have emi-grated out of the lymphoid tissues to the inflamed tissues, thisapparent lack of CD4� T cell expansion in the lymphoid tissues inIL-2R� KO mice differs from IL-2 KO and IL-2R� KO mice (8,12, 14, 15). Whether this is due to the intrinsic property of IL-2R�targeted mutation remains to be determined.

Interestingly, there is a significant increase in CD4� T cells inthe bone marrow (from 1.3 � 106 in femurs in B6 mice to 3 � 106

in IL-2R� KO mice and 4 � 106 in DM mice). In contrast tonormal B6 control, a high proportion of the CD4� T cells in thebone marrow of IL-2R� KO and DM mice expressed CD69 (datanot shown). This may explain why a 2- to 3-fold increase in CD4�

T cells is sufficient to induce disease phenotype in IL-2R� KO andDM mice. Alternatively, the bone marrow is highly sensitive toinflammation-mediated effects of activated T cells. The reason forthe accumulation of CD4� T cells is unclear at present, but it isconsistent with the marked abnormalities observed in the bonemarrow and the importance of CD4� T cells to induce the diseasephenotypes as demonstrated by anti-CD4 mAb blocking and adop-tive transfer experiments (14).

Table I. Numbers and percentages of B and T cell in the lymph nodes (LN) and spleens (Sp) of B6, IL-2R� KO, and DM micea

4 wk 10 wk 22 wk

B220 Thy-1 B220 Thy-1 B220 Thy-1

B6LNb % 28 6 69 5 36 10 54 11 58 12 35 13

Cells (106) 3 1 6 1 3 1 5 1 4 1 3 1Sp % 54 13 24 7 51 3 31 10 63 13 30 13

Cells (106) 38 9 17 5 42 2 26 8 59 12 28 12

IL-2R� KOc

LNb % 21 3 63 4 17 1 72 2 11 � 3 78 � 7Cells (106) 5 1 15 1 5 1 22 1 2 � 1 17 � 2

Sp % 30 4 47 7 27 4 64 6 10 � 3 63 � 6Cells (106) 28 3 44 6 32 7 76 7 12 � 4 72 � 7

DMLNb % 24 10 69 9 19 3 69 3 4 3 87 9

Cells (106) 5 2 14 2 4 1 16 1 1 0.5 24 3Sp % 33 15 39 10 19 10 66 6 9 5 72 13

Cells (106) 30 13 35 9 24 13 83 7 12 7 101 18

a Viable cells were counted by trypan blue dye exclusion. Proportions were determined by flow cytometry and absolute numbers were calculated. Loss of cells due to washingand filtering was not factored in. The values represent mean SD from at least three mice per group.

b Axillary, inguinal, and posterior cervical LN were collected and analyzed.c Values in bold were obtained from 14- to 16-wk-old IL-2R� KO mice that survived the early mortality associated with this strain.

Table II. Comparison of CD4� T cell numbers obtained from lymphnodes (LN), spleen (Sp), and bone marrows (BM) of B6, IL-2R� KO,and DM micea

CD4� T Cells (�106)

4 wk 10 wk 22 wk

B6LNb 4 1 4 1 2 0.5Sp 12 2 20 1 18 2BMc 1.1 0.1 0.7 0.2 1.3 0.4

IL-2R� KOd

LNb 5 1 5 1 2 � 0.4Sp 15 2 24 3 23 � 6BMc 2 0.4 3 1 3 � 0.5

DMLNb 4 1 4 1 3 1Sp 15 3 24 4 36 9BMc 1.3 0.3 3 0.2 4 1

a Cells were analyzed as described in Table I. Values represent mean SD fromat least three mice per group.

b Axillary, inguinal, and posterior cervical lymph nodes were collected andanalyzed.

c Bone marrow cells were obtained from femurs of individual mice.d Values in bold were obtained from 14- to 16-wk-old IL-2R� KO mice that

survived the early mortality associated with this strain.

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DiscussionThe consequence of the immune defect in IL-2R� targeted muta-tion is very complex. Recent advance in Treg cell research stronglysuggests that many of the various immune phenotypes observed inIL-2R� KO mice are initiated by the lifelong absence of theCD4�CD25� Treg cells. In the absence of this population of Tregcells, lymphocytes in young mice are polyclonally activated andcertain tissues are infiltrated by these activated lymphocytes, lead-ing to tissue inflammation and pathology. We characterized theinflammatory responses and the Fas-based tissue damage in vari-ous tissues/organs. Among them, the lung and colon pathology aresevere and may contribute to mortality. Importantly, we also foundthat bone marrow is a major target of infiltration by activated Tcells and certain immune phenotypes observed in the IL-2R� KOmice is likely the consequence of this process. We demonstratedthat along with bone marrow infiltration of activated T cells,IL-2R� KO mice developed anemia and blocked the formationof pre-B/immature B cells, the latter event was followed by Bcell depletion in peripheral lymphoid tissues and the reductionof serum IgG level in aged mice. Our study covers the lifespanof the mice starting from genotyping at 4 wk of age to the pointwhere the mice are moribund. A schematic presentation of thesepathways/processes is shown in Fig. 10, which describes ourview on the immune system biology in IL-2R� KO mice basedon the roles of Treg cells and Fas in this system (see figurelegend). This hypothesis is supported by the fact that these phe-notypes are progressive and age-dependent and that similar phe-notypes were observed in IL-2 KO mice and IL-2R� KO micethat also lack the CD4�CD25� Treg cells (8, 10, 12, 14). Ourdata also demonstrated the dominant feature of Treg cell ab-normality over Fas mutation because the majority of the phe-notypes in the DM mice resemble IL-2R� KO mice (multiorganinflammation and early increase in serum IgG, etc.) but not theB6.lpr mice (accumulation of the abnormal double-negative Tcells (data not shown) and late increase in serum IgG level).

B cell deletion has been reported in a few old IL-2 KO mice(9, 12) and IL-2R� KO mice (14) but not IL-2R� KO mice (13).B cell deletion in IL-2 KO mice requires T cells because it wasnot observed in nude IL-2 KO mice, and B cells in nude micewere deleted upon adoptive transfer of T cells from IL-2 KOmice (12). B cell deletion in IL-2R� KO mice requires CD4� Tcells because it was prevented by anti-CD4 mAb treatment (14).In both IL-2 KO mice and IL-2R� KO mice, the differentiationstep from pro-B cells to pre-B/immature B cells in the bonemarrow was blocked (12, 14). This blockade was demonstratedin IL-2R� KO mice in the present study. However, B cell de-velopment was apparently normal in mice before 4 – 8 wk ofage, arguing against the interpretation that IL-2/IL-2R signalingis essential for B cell development (12). Collectively, thesestudies suggest that the autoimmune expansion of CD4� T cellsmust have occurred and they are responsible for the B cell de-letion. It was hypothesized that these activated T cells infiltratebone marrow and interfere with the B cell differentiation pro-cess (12). We showed that in IL-2R� KO mice, the absolutenumber of CD4� T cells infiltrated in bone marrow was sig-nificantly increased, providing strong supporting evidence thatinfiltration of lymphocytes into the bone marrow is affecting thegeneration of pre-B/immature B cells. A number of mechanismshave been suggested, including T cell-mediated killing of Bcells (12, 24). Our study demonstrates that the bone marrow Bcell deletion phenotype in IL-2R� KO mice is Fas-independentbecause the differentiation from pro-B cells to pre-B/immatureB cells is also observed in the DM mice. Moreover, the absolutenumber of B cells in the peripheral lymphoid tissues was re-duced significantly both in old IL-2R� KO and DM mice. Theseobservations indicate that B cell depletion in the periphery ismostly Fas-independent. They also suggest that the B cell dif-ferentiation block in the bone marrow is the main mechanismresponsible for the peripheral B cell depletion in older mice.

We also observed a remarkable increase in CD8� T cells in theIL-2R� KO and DM mice. This phenotype is not observed in IL-2KO mice (data not shown) and not reported in IL-2R� KO mice (14).This phenotype is not described in detail in this study because it isintrinsic to IL-2R� KO and independent from Treg expression defect.In IL-2R� KO mice, the IL-2R/IL-15R �-chain expression is up-regulated (25) and the �� chains are more available for pairing withIL-15R� to form the high affinity IL-15R. The overexpression of highaffinity IL-15R and the omnipresence of IL-15 in virtually all tissues(26, 27) could provide a favorable condition for T cell expansion.Additionally, IL-15/IL-15R interaction appears to affect preferentiallyCD8� T cell homeostasis by providing a strong antiapoptotic signalthat favors CD8� T cell survival (28). In support of this, there is a40–50% reduction in the CD8� T cell number in the lymph nodesand spleens of IL-15 KO and IL-15R KO mice compared with theirnormal counterparts (29, 30). Our observation of a remarkable in-crease in CD8� T cells in IL-2R� KO mice provides a reciprocalsituation to IL-15 KO mice and IL-15R� KO mice.

Increase in CD4� T cell numbers was not observed in the lym-phoid tissues of IL-2R� KO mice except in bone marrow. It islikely that many activated T cells have already infiltrated into var-ious inflamed tissues. Therefore, the apparent lack of an increase inCD4� T cells cannot be taken as evidence of lacking T cell acti-vation and expansion. In support of this interpretation, the propor-tion of activated phenotype, as defined by expression of activationmarker CD69, increased 2.5- to 4-fold in the lymphoid tissues ofIL-2R� KO and DM mice in comparison with B6 counterparts(Fig. 3). This is observed in both CD4� and CD8� T cell popu-lations. It is important to note that in the bone marrow of IL-2R�KO and DM mice, both the total number of CD4� T cells and the

FIGURE 10. Roles of Treg cells and Fas played in the immune sys-tem biology in IL-2R� KO mice: a schematic presentation. In newbornIL-2R� KO mouse, thymocyte differentiation produces CD4�CD25� Tcells but not CD4�CD25� Treg cells. In the periphery, CD4�CD25� Tcells are spontaneously and polyclonally activated and expanded in theabsence of Treg cells. Some of the autoimmune T cells go to varioustissues/organs and cause inflammation and Fas-based tissue damages.Some autoimmune T cells go to bone marrow where they cause bonemarrow depression, leading to inhibition of erythropoiesis and B celllymphopoiesis that contribute, respectively, to anemia and B cell dele-tion. We have not yet determined whether the generation of thymocyteprecursors is inhibited.

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percentage of activated phenotype of CD4� T cells were increasedin comparison to B6 control, consistent with the interpretation thatthese infiltrated T cells are responsible for the suppression of bonemarrow function observed in these mice.

The inflamed tissue studied most in IL-2 KO, IL-2R� KO, andIL-2R� KO mice is colon, and ulcerative colitis has been consideredas a major cause and predictor for mortality. We have previouslyshown that lpr mutation does not inhibit colon inflammation but in-hibits colon epithelial cell apoptosis and colon damage in IL-2 KOmice (21). We attributed this protection of colon damage to the pre-vention of mortality. The data presented in the present study indicateinflammation in multiple tissues so that attribution of colitis to mor-tality may be simplistic at least in the case of IL-2R� KO mice. Par-ticularly, the damage in the lung is so severe that it is likely to con-tribute to the mortality observed in IL-2R� KO mice. A severeinflammation in the lung as well as several phenotypes describedherein were also reported for a patient bearing a deletion mutant IL-2R� gene (31). Inflammation in lung, pancreas, and colon was alsoreported in gnotobiotic IL-2 KO mice (10). Despite the protection oftissue cell apoptosis by Fas mutation, mortality was only protectedpartially. Thus, mortality in IL-2R� KO mice involves both Fas-de-pendent and Fas-independent mechanisms.

In summary, we have characterized in great detail a number ofphenotypes in IL-2R� KO mice and determined the role of Fasplayed in these phenotypes. In addition to colon, we have identi-fied new tissues that are inflamed and some that are damaged byFas-based mechanisms. Among them, we identified bone marrowas a target of autoimmune T cells that are expanded in the absenceof the CD4�CD25� Treg cells. Data presented suggest that B celldeletion and anemia are the consequences of the infiltration ofthese autoimmune T cells that suppress bone marrow function. Ofgreat significance is that we described many of these phenotypes ina time- and age-dependent fashion, thus, providing a lifelong andglobal picture of the immune system biology of IL-2R� KO mice.In this view, the effect of Treg cells is largely independent fromFas mutation because FasL induction and Fas up-regulation occurssubsequent to the polyclonal T cell activation resulted from life-long absence of the CD4�CD25� Treg cells. Accordingly, the roleof Fas on the immune system biology of IL-2R� KO mice be-comes evident only in older mice when the autoimmune inflam-mation is fully developed. Indeed, Fas mutation did not block in-flammation of various organs and only affected phenotypes ofinflammation that are Fas-dependent such as apoptosis and che-motaxis. The immune system biology in IL-2R� KO mice, as de-picted in Fig. 10, is based on the loss of the CD4�CD25� Tregcells and through information gathered from both in vitro and invivo research. This scheme provides a general map for future stud-ies and for the refinement of the immune system biology in micelacking CD4�CD25� Treg cells.

DisclosuresThe authors have no financial conflict of interest.

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