The Role of Fas in the Biology of IL-2R α Knockout Mice: Interplay among Regulatory T Cells, , Hemopoiesis, and Apoptosis This information is current as of September 26, 2021. Rahul Sharma, Harini Bagavant, Wael N. Jarjour, Sun-Sang J. Sung and Shyr-Te Ju J Immunol 2005; 175:1965-1973; ; doi: 10.4049/jimmunol.175.3.1965 http://www.jimmunol.org/content/175/3/1965 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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 the role of Fas in the immune system biology of IL-2R␣ KO mice. Consistent with a defect in CD4؉CD25؉ regulatory T (Treg) cell expression, spontaneous 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 -infiltrated bone marrow, lymphopoiesis was blocked at pro-B to pre-B/immature B stage, culminating in an age-dependent B cell loss in the periphery. These phenotypes were also observed in IL-2R␣ KO mice bearing the lpr mutation (DM mice), indicating Treg cell function and the phenotypes attributed Downloaded from directly 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 these processes. Our age-dependent, lifelong analysis of IL-2R␣ KO and DM mice supports a CD4؉CD25؉ Treg cell-based mechanism for the abnormal immune system biology observed in IL-2R␣ KO mice and provides a global view of the interplays among Treg cells, multiorgan inflammation, hemopoiesis, and apoptosis. The Journal of Immunology, 2005, 175: 1965–1973. http://www.jimmunol.org/ he IL-2/IL-2R signaling pathway plays a critical role in letion, tissue inflammation, tissue cell death (other than colon) was the immune system (reviewed in Refs. 1–3). It is a pow- not determined (21). erful signal for lymphocyte proliferation and a critical It is likely that a common mechanism is responsible for many of T ϩ ϩ factor for the development and expansion of the CD4 CD25 the phenotypes shared among IL-2 KO, IL-2R␤ KO, and IL-2R␣ regulatory T (Treg)2 cells that down-regulate proliferation/ KO mice. We propose a hypothesis based on a current concept of activation. A characteristic phenotype of Treg cell absence is tissue Treg cell function to explain the shared phenotypes among these inflammation (4–7). Targeted mutation of IL-2, IL-2R␣ (CD25), mouse strains. We hypothesize that the absence of Treg cells al- or IL-2R␤ (CD122) gene results in a similar phenotype character- lows CD4ϩCD25Ϫ T cells (including autoimmune T cells) to be ized not only by tissue inflammation as exemplified by ulcerative polyclonally activated. Activated autoimmune T cells infiltrate by guest on September 26, 2021 colitis but also by severe anemia, body weight loss, and other specific target organs where they interfere with organ functions, immune abnormalities such as B cell deletion reported in old IL-2 leading to various phenotypes. In addition, autoimmune T cells knockout (KO) mice and IL-2R␤ KO mice (8–15). A consequence that target bone marrow are induced, and bone marrow is highly associated with these abnormalities is early mortality, which pre- sensitive to these cells. T cell infiltration into bone marrow sup- vents a thorough analysis of the immune system defect and im- presses bone marrow function, leading to progressive hemopoie- mune regulation in these mice, especially in adult and older mice. sis-based abnormalities. We choose to study IL-2R␣ KO mice be- The FasL (CD178)/Fas (CD95)-mediated apoptosis pathway is cause T cell infiltration into bone marrow, B cell deletion, and critical to the maintenance of peripheral tolerance, most notably in inflammation in multiple organs were not described (13). Also, we systemic such as the lupus-like autoimmune introduced lpr mutation into IL-2R␣ KO mice (referred to as DM disease in lpr and gld mice (16, 17). In addition, FasL on infil- mice) to determine whether a specific phenotype is dependent on trating lymphocytes of the inflamed tissues have been shown to Treg cells or Fas expression. This is significant because Fas mu- induce apoptosis of tissue cells (18–21). We recently have shown tation prolonged the lifespan of IL-2R␣ KO mice. Fas mutation did that introducing the lpr mutation into IL-2 KO mice prolongs their not prevent inflammation in multiple organs. However, it affected lifespan (21). This is attributed at least in part to the prevention of the pathology of the inflamed lung and colon. Like IL-2 KO and anemia and in part to the prevention of the FasL-induced apoptosis IL-2R␤ KO mice, we also observed T cell infiltration into bone of colon epithelial cells. However, whether Fas-mediated signaling marrow where erythropoiesis and B cell lymphopoiesis were im- pathways are involved in a specific phenotype such as B cell de- 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 throughout *Division of Rheumatology and Immunology, Department of Internal Medicine, Uni- the lifespan of IL-2R␣ KO mice by characterizing their specific versity of Virginia, Charlottesville, VA 22908 immune phenotypes and by defining the roles of CD4ϩCD25ϩ Received for publication February 28, 2005. Accepted for publication May 5, 2005. Treg cells and Fas in these phenotypes. 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. Materials and Methods Breeding and genotyping 1 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Division of Rheu- matology and Immunology, Department of Internal Medicine, University of Virginia, C57BL/6.Il2R␣ϩ/Ϫ mice and B6.MRL-Faslpr/J (B6.lpr or lprϩ/ϩ) mice Charlottesville, VA 22908-0412. E-mail address: [email protected] were obtained from The Jackson Laboratory. Because Il2R␣Ϫ/Ϫ (IL-2R␣ ϩ Ϫ ϩ ϩ 2 Abbreviations used in this paper: Treg, regulatory T; KO, knockout. KO) mice are sterile, Il2R␣ / mice were first bred with lpr / mice to

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1966 Treg CELL- AND Fas-REGULATED IMMUNE PHENOTYPES

␣ϩ/Ϫ ϩ/Ϫ obtain Il2R lpr F1 offspring. F1 mice were intercrossed to obtain Il2R␣Ϫ/Ϫlprϩ/ϩ and Il2R␣ϩ/Ϫlprϩ/ϩ mice. We then intercrossed Il2R␣ϩ/ ϩ ϩ Ϫ Ϫ ϩ ϩ Ϫlpr / mice to increase the frequency of Il2R␣ / lpr / offspring. Tail DNA 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 and reaction conditions from The Jackson Laboratory website (͗www.jax.org͘). PCR products were determined by their sizes as follows: 218 bp for fas mutant of lpr mice, 182 bp for the normal fas, 280 bp for the Il2R␣ mutant allele, and 146 bp for the normal Il2R␣ allele. DNA samples from C57BL/6J (B6), IL-2R␣ KO, and B6.lpr mice were used as controls. Mice were housed at the University of Virginia animal facility, and experiments were 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. Ten microliters of blood samples were collected and immediately diluted in 90 ␮l of cold PBS, pH 7.2. Aliquots were used to determine hemoglobin levels and cell-free samples were collected by centrifugation and used for the determination of IgG levels using the mouse IgG ELISA kit (Bethyl Downloaded from Laboratories). Determination of hemoglobin level Blood samples were diluted 100-fold with dilution buffer (3 mM potassium ferricyanide, 1.5 mM potassium cyanide, 5 mM sodium borate, 0.1% Non- idet P-40) in which RBC were lysed and the released hemoglobin was converted to cyanomethemoglobin. The relative hemoglobin concentra- tions were determined photometrically at 546 nm. http://www.jimmunol.org/ Immunohistochemical and immunofluorescence staining ␣ Sections of paraffin-embedded tissues were stained with H&E. Tissues ex- FIGURE 1. Genotyping and phenotyping IL-2R KO and DM mice. A, amined are lungs, thyroids, lacrimal glands, salivary glands, hearts, stom- The PCR product sizes are 218 and 182 bp for the lpr and wild-type fas achs, livers, pancreas, kidneys, small intestines, colons, and ovaries. Ap- allele, respectively. The PCR product sizes for the mutant and wild-type optosis was determined using a TUNEL assay kit (Apoptag Plus IL-2R␣ are 280 and 146 bp, respectively. In this example, lane 1 and lane Peroxidase In Situ Apoptosis Detection kit; Serologicals). We followed the 8 represent DM mice, lane 3 has the IL-2R␣ KO genotype, lane 5 is lpr, manufacturer’s protocol and included the positive control provided by the and lane 7 is a wild-type mouse. Lane 6 is molecular size standard. B, manufacturer. Background staining was determined by incubation with Lymph node cells of genotyped mice were stained with FITC-anti-Fas by guest on September 26, 2021 buffer in the absence of TdT. Slides were counterstained with methylene mAb for Fas expression and analyzed with flow cytometry (shaded, iso- blue followed by graded alcohol dehydration and mounting. TUNELϩ cells type; thin line, DM; thick line, B6). C, The genotyped DM mouse is neg- developed a characteristic brown/black color in the nuclear region. ative for CD4ϩCD25ϩ Treg cells. 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- mAb for Fas expression and FITC-anti-CD4 plus PE-anti-CD25 sciences). After washing, slides were incubated for 30 min with Alexa mAb for Treg cells (Fig. 1, B and C). Mice were observed weekly 647-conjugated goat anti-hamster Ab (Molecular Probes). For a negative for general appearance and mortality. In addition, body weight control, we used rat IgG and hamster IgG (both from BD Biosciences) in gain/loss and the severity of anemia were determined. The com- place of anti-B220 mAb and anti-CD3 mAb, respectively. The slides were ␣ examined using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss). piled data are shown in Fig. 2. Mice bearing both Fas and IL-2R - targeted mutations (DM mice) survived significantly longer than Flow cytometric analysis IL-2R␣ KO mice but did not reach to the life span of B6.lpr and Lymphocytes in blood, lymph nodes, spleen, bone marrow, and thymus B6 mice (Fig. 2A). Severe anemia as defined by Յ50% of control were analyzed. Single cell suspensions of samples were treated with am- B6 hemoglobin level was observed in 10-wk- but not 4-wk-old monium chloride to remove erythrocytes, washed, and then stained for IL-2R␣ KO and DM mice (Fig. 2B). Interestingly, the protection 6 ␮ various cell surface markers. Cells (10 ) were suspended in 100 lofPBS of anemia by lpr mutation in DM mice is moderate for males and containing 4% BSA and incubated with 1 ␮g of various fluorescent Abs for 30 min at 4°C. FITC- and/or PE- conjugated anti-CD4 (H129.19), anti- unremarkable for females (Fig. 2B). The reason for the differential CD8 (53-6.7), anti-CD25 (PC61), anti-Thy-1.2 (30H12), anti-B220 (RA3- protection is unclear. A moderate protection of body weight loss as 6B2), anti-CD43 (S7), anti-Fas (Jo-2), and anti-CD69 (H1.2F3) mAb were compared with IL-2R␣ KO mice was observed in male DM mice obtained from BD Biosciences. FITC conjugated goat anti-mouse IgM was but not in the female DM mice (Fig. 2C). Still, Fas mutation pro- purchased from Southern Biotechnology Associates. At least 104 stained cells were analyzed using a FACScan (BD Biosciences) equipped with longed the lifespan of female mice as well as male mice. The CellQuest (BD Biosciences). Post acquisition analyses were conducted us- partial and marginal improvement of body weight gain and the ing FlowJo software (Tree Star). weak prevention of anemia agree with the inability of these mice to reach the lifespan of B6.lpr and B6 mice. Results Fas mutation prevents early mortality and prolongs the lifespan Spontaneous T cell activation in 6-wk-old IL-2R␣ KO of IL-2R␣ KO mice and DM mice We introduced the fas mutant gene of B6.lpr mice into IL-2R␣ KO We determined whether spontaneous T cell activation occurs as a mice by breeding. The genotypes of progeny were identified by result of lifelong absence of the CD4ϩCD25ϩ Treg cells. As PCR on tail DNA samples (Fig. 1A). The cell surface phenotypes shown in Fig. 3, when compared with age- and sex-matched B6 were demonstrated by staining lymphocytes with FITC-anti-Fas mice, a significantly higher proportion of the CD4ϩ T cells in the The Journal of Immunology 1967

FIGURE 3. Spontaneous activation of T cells in IL-2R␣ KO and DM mice. Lymph node cells from 6-wk-old female B6, IL-2R␣ KO, and DM mice were stained with anti-CD4 mAb plus anti-CD69 mAb (upper panels) and

anti-CD8 mAb plus anti-CD69 mAb (lower panels). Please note that the num- Downloaded from bers in the quadrants are percentages of the population of total lymphocytes.

lar lumen along with hypertrophy of the lining epithelium. The reason why infiltration was observed in the lung of some but not all IL-2R␣ KO mice is not clear at present but its

presence is consistent with the idea that FasL is a potent chemo- http://www.jimmunol.org/ tactic factor for (22). The colonic mucosa in both IL-2R␣ KO and DM mice showed significant thickening and hypertrophy compared with control B6 FIGURE 2. Fas mutation protects IL-2R␣ KO mice from early mortality, mice (Fig. 4). Lymphocytic infiltration in the lamina propria was ob- anemia, and body weight loss. The DM mice lived longer than IL-2R␣ KO served in both groups but was more prominent in the DM mice. mice (A) and had improved blood hemoglobin levels (B, male; C, female) and Multinucleated giant cells were also observed in the colonic mucosa body weight (D, male; E, female). The number of mice examined are equal to of IL-2R␣ KO mice. The submucosa and muscular layers appeared or more than six (n Ն 6) except for IL-2R␣ KO mice (n ϭ 5 for male and n ϭ normal. 3 for female). Changes in the pancreas and lacrimal glands were characterized by guest on September 26, 2021 by extensive lymphocytic infiltration in the periductal regions. There was little involvement of the exocrine glands. In the pan- lymph nodes of 6-wk-old IL-2R␣ KO (6 of 12 or 50%) and DM (7 creas, infiltration did not appear to invade the pancreatic islets or of 18 or 39%) mice expressed the activation marker CD69, cause islet cell destruction. These mice did not develop diabetes whereas in B6 mice the value was 16%. Similarly, increase in mellitus as determined by urinary glucose level (data not shown). CD69 expression was observed for CD8ϩ T cells. We noted that Similar periductal infiltration was seen in salivary glands of both there was an increase in CD8ϩ T cells in both IL-2R␣ KO and DM IL-2R␣ KO and DM mice. Inflammation in thyroid, liver, stom- mice (data not shown) and the proportion of CD69ϩ cells within ach, small intestine, ovary, kidney, and heart was either very mild the CD8ϩ T cell population was somewhat less than the CD69ϩ or not observed (data not shown). No pathological changes were cells in the CD4ϩ T cell population. apparent in tissues from age-matched control B6 and B6.lpr mice. These data suggest that inflammation in multiple tissues/organs is Fas mutation did not prevent tissue inflammation due to the lack of CD4ϩCD25ϩ Treg cells in both IL-2R␣ KO and We determined whether IL-2R␣ KO mice (3–4 mo old) develop DM mice. In addition, Fas deficiency in DM mice influences the inflammatory response in multiple tissues/organs following the qualitative nature (infiltrating cell type) and extent of inflammation spontaneous T cell activation. Various tissues were collected from (colon in DM mice) of the inflammatory response. B6, B6.lpr, IL-2R␣ KO, and DM mice and the stained tissue slides were examined. As shown in Fig. 4, IL-2R␣ KO and DM mice Fas mutation inhibited apoptosis of tissue cells in the inflamed showed histopathological changes in lungs, colon, pancreas, and lung and colon lacrimal glands in comparison to the counterparts of B6 and B6.lpr To determine whether tissue cells in the severely inflamed sites (data not shown) mice. Both IL-2R␣ KO and DM mice developed were killed by Fas/FasL-mediated apoptosis, we used the TUNEL lung inflammation. The severity of inflammation was greater in the assay to determine the cell types and the number of apoptotic cells IL-2R␣ KO mice compared with DM mice. Both showed cellular in the lung and colon of IL-2R␣ KO and DM mice. A represen- infiltrates surrounding the bronchiolar lumen extending into the tative experiment is shown in Fig. 5. Few apoptotic cells were lung parenchyma. However, there were differences in the nature of observed in the lung of a B6 mouse. Many apoptotic cells were the infiltrating cells. Lymphocytes were the major infiltrating cells observed in the lung of the 16-wk-old IL-2R␣ KO mouse. Al- in all six DM mice examined. In three of the six IL-2R␣ KO mice though many of them were leukocytes, apoptotic tissue cells were examined, lymphocytes were also the major cell type in the af- evident (inset and right panels). In the lung of an age-matched DM fected regions. However, strong infiltration of both neutrophils and mouse, the number of apoptotic leukocytes was significantly re- lymphocytes were observed in the other three mice. In some areas, duced in comparison with IL-2R␣ KO mouse. In addition, apo- the neutrophil-containing exudates formed a plug in the bronchio- ptotic tissue cells were rarely observed (inset and right panels). 1968 Treg CELL- AND Fas-REGULATED IMMUNE PHENOTYPES

FIGURE 4. Histological examina- tion of tissues from B6, IL-2R␣ KO, and DM mice. Various tissues were fixed in 10% neutral buffered forma- lin, paraffin embedded, and sectioned, and 4-␮m sections were stained with H&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- Downloaded from 2R␣ KO and DM mice is indicated. IL-2R␣ KO mice have a strong neu- trophil infiltration (arrows) in the lungs and bronchiolar lumen exudate (B). Arrow in inset in E shows a multinucleated giant cell seen in the http://www.jimmunol.org/ colonic mucosa of IL-2R␣ KO mice. Photomicrographs were taken at ϫ200 for lung (A–C) and colon (D– F). All other photomicrographs were taken at ϫ100 magnification. Little or no infiltration was observed in tissues of control B6 mouse. by guest on September 26, 2021

Similarly, a higher number of apoptotic cells were detected in the in Fig. 6. The percentages of B220ϩIgMϪ pre-B/immature B cells inflamed colon of IL-2R␣ KO mice (Fig. 5). Both apoptotic cells (from 6.13% in B6 to 0.36% in IL-2R␣ KO and 1.03% in DM and tissue damage were less evident in the inflamed colon of DM mice) and B220ϩIgMϩ mature B cells (from 3.01% in B6 to mouse than IL-2R␣ KO mouse. These observations suggest that 0.33% in IL-2R␣ KO and 0.58% in DM mice) were greatly re- FasL is induced in IL-2R␣ KO mice and that Fas-mediated apo- duced in the bone marrow of IL-2R␣ KO and DM mice, whereas ptosis plays a major role in cell death during the inflammatory the percentage of B220ϩCD43ϩ pro-B/small pre-B cells was not ␣ response in IL-2R KO mice. significantly different from a B6 control (4.47% in B6 vs 3.69% in ␣ IL-2R␣ KO mice display Fas-independent, age-dependent IL-2R KO and 4.75% in DM mice). The data indicate that bone deletion of B cells marrow B cell differentiation is blocked at the pro-B to pre-B/ immature B cells stage and this phenotype is mostly Fas-indepen- Blockade of B cell differentiation in bone marrow. Previous dent. It is important to note that the total number of bone marrow studies of IL-2 KO and IL-2R␤ KO mice have shown that B cell cell count (after depletion of erythrocytes) was comparable among lymphopoiesis was severely blocked at the pro-B to pre-B/imma- these mice (data not shown), indicating that the output of naive B ture B cell stage as a result of infiltration of lymphocytes and cells into the periphery is greatly reduced in the old IL-2R␣ KO leukocytes, respectively (12, 14). We also observed a strong infil- and DM mice as a consequence of this blockade. tration of T cells in the bone marrow of IL-2R␣ KO and DM mice (see Table III). Therefore, we examined B cell lymphopoiesis in Depletion of B cell zone in lymph nodes. We reasoned that a the bone marrow of both the IL-2R␣ KO (16 wk old) and DM (22 direct consequence of lacking an input of B cells from bone mar- wk old) mice using anti-B220, anti-IgM, and anti-CD43 mAb to row could result in B cell depletion in the periphery. Following the determine the B cell subpopulations at various differentiation immature B cell differentiation blockade in the bone marrow, the stages in the bone marrow. A representative experiment is shown area of B cell zone in the lymph nodes of 14-wk-old mice was The Journal of Immunology 1969 Downloaded from http://www.jimmunol.org/ 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. TUNELϩ cells as indicated by dark brown color were more frequent in the IL-2R␣ KO mice than DM mice (ϫ100). The inset captured at ϫ400 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 TUNELϩ tissue cells, TUNELϩ 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 ϫ200. The tissue apoptotic cells were distinguished from apoptotic lymphocytes on the basis of the size of the nucleus and surrounding cytoplasm. proportionally reduced as demonstrated by two-color staining us- The age-dependent elevation of IgG followed by the dramatic de- ing anti-B220 mAb/anti-CD3 mAb and examined with a confocal cline in the sera of IL-2R␣ KO and DM mice is consistent with the by guest on September 26, 2021 microscope (Fig. 7). This reduction is significant considering the hypothesis that absence of Treg cells in these mice permitted an early fact that the lymph node size of IL-2R␣ KO and DM mice was T cell-dependent polyclonal activation of B cells, and a subsequent much larger than B6 control. In addition, a more severe B cell zone deletion of B cells resulted from T cell infiltration into bone marrow. depletion was observed in 22-wk-old DM mice (data not shown). In addition, the deranged lymphoid architecture in these mice may not Interestingly, the remaining B cell zone was infiltrated with T cells. Infiltration of T cells into B cell zone and loss of T/B cell demarcation was also observed in the B6.lpr lymph node as pre- viously reported (23). Consequently, both Treg cell abnormality and Fas mutation could contribute to the T cell infiltration into B cell 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 polyclonal lymphocyte activation and the subsequent B cell deletion should affect serum IgG levels. Therefore, the total serum IgG levels of mice at various ages were determined (Fig. 8). The IgG level of B6 mice did not fluctuate much during the entire period of study. As early as 1 mo old, the IgG level of IL-2R␣ KO mice was already significantly higher 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. The IgG 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- FIGURE 6. B cell development arrest in the bone marrow of IL-2R␣ dependent pattern of IgG expression level in the DM mice resembles KO mice is Fas-independent. Bone marrow cells from B6, IL-2R␣ KO, and that of the IL-2R␣ KO mice but not the B6.lpr mice (Fig. 8). Espe- DM mice were stained with PE-anti-B220 mAb and FITC-anti-CD43 mAb. cially, the increase in IgG level in old B6.lpr mice was not observed B cell development in bone marrow was affected at pre-B/immature B cell stage. Both B220highIgMϩ (mature B cells) and B220intIgMϪ (immature B in the old DM mice. The serum IgG level in the 22-wk-old DM mice cells) cells were reduced in IL-2R␣ KO and DM mice (top row). The dropped to 30% of the level of B6 mice at 9 mo of age (Fig. 8). The B220ϩCD43Ϫ cells (late pre-B, immature B and mature B cells) were data demonstrated a dominant role of Treg cell abnormality over Fas affected in both IL-2R␣ KO and DM mice, whereas the B220ϩCD43ϩ mutation in regulating serum IgG level. This dominance is consistent (pro-B and early pre-B) population was not (bottom row). The B6 and DM with the early lymphocyte activation and the late B cell deletion in the mice were 22 wk old while the IL-2R␣ KO mouse was 16 wk old. A IL-2R␣ KO and DM mice. representative of three experiments is shown. 1970 Treg CELL- AND Fas-REGULATED IMMUNE PHENOTYPES Downloaded from http://www.jimmunol.org/ 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␣ KO and 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 goat anti-hamster Ab, respectively (top panels) and examined under a confocal microscope. All mice used in this experiment were 14 wk old. Note the lymph node 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 zones and infiltration of T cells into the B cell zones were observed for B6.lpr, IL-2R␣ KO, and DM mice. allow optimal T/B interaction and this may also contribute to the of B cells in the lymphocyte population in blood was moderately age-dependent decline in serum IgG level. Finally, the slower rate of reduced (70% of B6 control). This value rapidly declined to a plateau decline of the serum IgG level in the DM mice in comparison to of 26% in 10-wk-old mice and 28% in 22-wk-old mice. by guest on September 26, 2021 IL-2R␣ KO mice suggests a small role of Fas mutation in this phe- To determine the actual changes in B cell number and propor- notype by protecting lymphocytes from FasL-mediated apoptosis. tion in lymph nodes, spleen, and bone marrow, individual mice were euthanized at specific ages and single cell suspensions were Changes in peripheral B cell numbers. prepared and counted as described in Materials and Methods. Cells Because we also observed changes in B cell proportion in the blood were stained with PE-anti-B220 and FITC-anti-Thy-1 mAb, and samples of IL-2R␣ KO and DM mice, the age dependence of this phenotype was determined for individual mice by staining blood sam- ples obtained at various age points with anti-B220 and anti-Thy-1 mAb (Fig. 9). At 4 wk of age, the earliest age analyzed, the percentage

FIGURE 8. Age-dependent changes in serum IgG levels. Serum sam- ples from individual mice (n ϭ 3) obtained at specific age were pooled. IgG levels were determined by ELISA as described in Materials and Methods. The pattern of IgG levels of DM mice resembles that of IL-2R␣ KO mice FIGURE 9. IL-2R␣ KO and DM mice display a progressive, age-de- but not B6.lpr mice. The IL-2R␣ KO and DM mice had very high serum pendent reduction of B cells in the blood. Blood samples of individual B6, IgG levels at a young age that progressively declined with age. Early mor- IL-2R␣ KO, and DM mice were obtained at various age points and stained tality prevented sample procurement from IL-2R␣ KO mice Ͼ4 mo old. with PE-anti-B220 and FITC-anti-Thy-1.2. IL-2R␣ KO mice did not sur- The data is a representative of three similar experiments. vive for the 22-wk sampling. The Journal of Immunology 1971

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

4wk 10wk 22wk

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

B6 LNb %28Ϯ 669Ϯ 536Ϯ 10 54 Ϯ 11 58 Ϯ 12 35 Ϯ 13 Cells (106) 3 Ϯ 16Ϯ 13Ϯ 15Ϯ 14Ϯ 13Ϯ 1 Sp % 54 Ϯ 13 24 Ϯ 751Ϯ 331Ϯ 10 63 Ϯ 13 30 Ϯ 13 Cells (106) 38 Ϯ 917Ϯ 542Ϯ 226Ϯ 859Ϯ 12 28 Ϯ 12

IL-2R␣ KOc LNb %21Ϯ 363Ϯ 417Ϯ 172Ϯ 2 11 ؎ 378؎ 7 Cells (106) 5 Ϯ 115Ϯ 15Ϯ 122Ϯ 1 2 ؎ 117؎ 2 Sp % 30 Ϯ 447Ϯ 727Ϯ 464Ϯ 6 10 ؎ 363؎ 6 Cells (106) 28 Ϯ 344Ϯ 632Ϯ 776Ϯ 7 12 ؎ 472؎ 7

DM LNb %24Ϯ 10 69 Ϯ 919Ϯ 369Ϯ 34Ϯ 387Ϯ 9 Cells (106) 5 Ϯ 214Ϯ 24Ϯ 116Ϯ 11Ϯ 0.5 24 Ϯ 3 Sp % 33 Ϯ 15 39 Ϯ 10 19 Ϯ 10 66 Ϯ 69Ϯ 572Ϯ 13 Cells (106) 30 Ϯ 13 35 Ϯ 924Ϯ 13 83 Ϯ 712Ϯ 7 101 Ϯ 18 Downloaded from

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 washing and filtering was not factored in. The values represent mean Ϯ 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.

the proportion was used to determine the total number of T and B due to early mortality), indicating that B cell deletion in the pe- http://www.jimmunol.org/ cells in each preparation. As shown in Table I, the relative per- riphery is mostly Fas-independent. We also observed a progressive, centages of B cell population in lymph nodes of 4-wk-, 10-wk-, age-dependent increase in Thy-1ϩ T cells in IL-2R␣ KO and DM and 22-wk-old DM mice were reduced, respectively, to 82% (24 of mice as compared with B6 control (Table I). This increase in Thy-1ϩ 28, p Ͼ 0.05), 53% (19 of 36, p Ͻ 0.05), and 7% (4 of 58, p Ͻ T cells is intrinsic to IL-2R␣ KO and is caused by a specific increase 0.05) of age-matched control mice. The absolute number of B cells in CD8ϩ T cells in IL-2R␣ KO and DM mice (data not shown). in the 4-wk- (5 ϫ 106 in DM vs 3 ϫ 106 in B6) and 10-wk-old (4 ϫ 106 in DM vs 3 ϫ 106 in B6) DM mice was not significantly ϩ different from age-matched B6 control. A significant decrease in Change in CD4 T cell numbers in bone marrow by guest on September 26, 2021 the absolute B cell number was observed only in 22-wk-old mice CD4ϩ T cells from IL-2 KO and IL-2R␤ KO mice have been ϫ 6 ϫ 6 (from 4 10 of B6 mouse to 1 10 of DM mouse), and this shown to transfer into nude mice several major phenotypes (11, 12, agreed well with the reduction of serum IgG level in these mice. 14). Therefore, we determined whether the absolute number of This pattern of B cell regulation is also observed in the spleen and CD4ϩ T cells was increased in these mice, we used FITC-anti- ␣ ϩ ϩ in IL-2R KO mice (4-, 10-, and 14-wk-old mice were analyzed CD4 mAb to stain CD4 T cells in the lymph nodes, spleen, and bone marrow of various mouse strains (Table II). For all lymphoid ϩ tissues studied except bone marrow and 22-wk-old DM mice, the Table II. Comparison of CD4 T cell numbers obtained from lymph ϩ nodes (LN), spleen (Sp), and bone marrows (BM) of B6, IL-2R␣ KO, total number of CD4 T cells was comparable to that observed in ϩ and DM micea the age-matched B6 controls, despite the fact that these CD4 T cells expressed activated phenotype based on CD69 expression ϩ CD4ϩ T Cells (ϫ106) (Fig. 3). Although some activated CD4 T cells must have emi- grated out of the lymphoid tissues to the inflamed tissues, this 4wk 10wk 22wk apparent lack of CD4ϩ T cell expansion in the lymphoid tissues in B6 IL-2R␣ KO mice differs from IL-2 KO and IL-2R␤ KO mice (8, b LN 4 Ϯ 14Ϯ 12Ϯ 0.5 12, 14, 15). Whether this is due to the intrinsic property of IL-2R␣ Sp 12 Ϯ 220Ϯ 118Ϯ 2 BMc 1.1 Ϯ 0.1 0.7 Ϯ 0.2 1.3 Ϯ 0.4 targeted mutation remains to be determined. Interestingly, there is a significant increase in CD4ϩ T cells in IL-2R␣ KOd the bone marrow (from 1.3 ϫ 106 in femurs in B6 mice to 3 ϫ 106 b LN 5 Ϯ 15Ϯ 1 2 ؎ 0.4 in IL-2R␣ KO mice and 4 ϫ 106 in DM mice). In contrast to Ϯ Ϯ 23 ؎ 6 ϩ Sp 15 2243 normal B6 control, a high proportion of the CD4 T cells in the BMc 2 Ϯ 0.4 3 Ϯ 1 3 ؎ 0.5 bone marrow of IL-2R␣ KO and DM mice expressed CD69 (data ϩ DM not shown). This may explain why a 2- to 3-fold increase in CD4 LNb 4 Ϯ 14Ϯ 13Ϯ 1 T cells is sufficient to induce disease phenotype in IL-2R␣ KO and Ϯ Ϯ Ϯ Sp 15 3244369 DM mice. Alternatively, the bone marrow is highly sensitive to BMc 1.3 Ϯ 0.3 3 Ϯ 0.2 4 Ϯ 1 inflammation-mediated effects of activated T cells. The reason for a Cells were analyzed as described in Table I. Values represent mean Ϯ SD from the accumulation of CD4ϩ T cells is unclear at present, but it is at least three mice per group. b Axillary, inguinal, and posterior cervical lymph nodes were collected and consistent with the marked abnormalities observed in the bone analyzed. marrow and the importance of CD4ϩ T cells to induce the disease 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 phenotypes as demonstrated by anti-CD4 mAb blocking and adop- survived the early mortality associated with this strain. tive transfer experiments (14). 1972 Treg CELL- AND Fas-REGULATED IMMUNE PHENOTYPES

Discussion B cell deletion has been reported in a few old IL-2 KO mice The consequence of the immune defect in IL-2R␣ targeted muta- (9, 12) and IL-2R␤ KO mice (14) but not IL-2R␣ KO mice (13). tion is very complex. Recent advance in Treg cell research strongly B cell deletion in IL-2 KO mice requires T cells because it was suggests that many of the various immune phenotypes observed in not observed in nude IL-2 KO mice, and B cells in nude mice IL-2R␣ KO mice are initiated by the lifelong absence of the were deleted upon adoptive transfer of T cells from IL-2 KO ϩ CD4ϩCD25ϩ Treg cells. In the absence of this population of Treg mice (12). B cell deletion in IL-2R␤ KO mice requires CD4 T cells, lymphocytes in young mice are polyclonally activated and cells because it was prevented by anti-CD4 mAb treatment (14). certain tissues are infiltrated by these activated lymphocytes, lead- In both IL-2 KO mice and IL-2R␤ KO mice, the differentiation ing to tissue inflammation and pathology. We characterized the step from pro-B cells to pre-B/immature B cells in the bone inflammatory responses and the Fas-based tissue damage in vari- marrow was blocked (12, 14). This blockade was demonstrated ous tissues/organs. Among them, the lung and colon pathology are in IL-2R␣ KO mice in the present study. However, B cell de- severe and may contribute to mortality. Importantly, we also found velopment was apparently normal in mice before 4–8 wk of that bone marrow is a major target of infiltration by activated T age, arguing against the interpretation that IL-2/IL-2R signaling cells and certain immune phenotypes observed in the IL-2R␣ KO is essential for B cell development (12). Collectively, these ϩ mice is likely the consequence of this process. We demonstrated studies suggest that the autoimmune expansion of CD4 T cells that along with bone marrow infiltration of activated T cells, must have occurred and they are responsible for the B cell de- IL-2R␣ KO mice developed anemia and blocked the formation letion. It was hypothesized that these activated T cells infiltrate of pre-B/immature B cells, the latter event was followed by B bone marrow and interfere with the B cell differentiation pro- cell depletion in peripheral lymphoid tissues and the reduction cess (12). We showed that in IL-2R␣ KO mice, the absolute Downloaded from ϩ of serum IgG level in aged mice. Our study covers the lifespan number of CD4 T cells infiltrated in bone marrow was sig- of the mice starting from genotyping at 4 wk of age to the point nificantly increased, providing strong supporting evidence that where the mice are moribund. A schematic presentation of these infiltration of lymphocytes into the bone marrow is affecting the pathways/processes is shown in Fig. 10, which describes our generation of pre-B/immature B cells. A number of mechanisms view on the immune system biology in IL-2R␣ KO mice based have been suggested, including T cell-mediated killing of B on the roles of Treg cells and Fas in this system (see figure cells (12, 24). Our study demonstrates that the bone marrow B http://www.jimmunol.org/ legend). This hypothesis is supported by the fact that these phe- cell deletion phenotype in IL-2R␣ KO mice is Fas-independent notypes are progressive and age-dependent and that similar phe- because the differentiation from pro-B cells to pre-B/immature notypes were observed in IL-2 KO mice and IL-2R␤ KO mice B cells is also observed in the DM mice. Moreover, the absolute that also lack the CD4ϩCD25ϩ Treg cells (8, 10, 12, 14). Our number of B cells in the peripheral lymphoid tissues was re- data also demonstrated the dominant feature of Treg cell ab- duced significantly both in old IL-2R␣ KO and DM mice. These normality over Fas mutation because the majority of the phe- observations indicate that B cell depletion in the periphery is notypes in the DM mice resemble IL-2R␣ KO mice (multiorgan mostly Fas-independent. They also suggest that the B cell dif- inflammation and early increase in serum IgG, etc.) but not the ferentiation block in the bone marrow is the main mechanism B6.lpr mice (accumulation of the abnormal double-negative T responsible for the peripheral B cell depletion in older mice. by guest on September 26, 2021 ϩ cells (data not shown) and late increase in serum IgG level). We also observed a remarkable increase in CD8 T cells in the IL-2R␣ KO and DM mice. This phenotype is not observed in IL-2 KO 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 is intrinsic 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 with IL-15R␣ to form the high affinity IL-15R. The overexpression of high affinity 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 preferentially CD8ϩ T cell homeostasis by providing a strong antiapoptotic signal that favors CD8ϩ T cell survival (28). In support of this, there is a 40–50% reduction in the CD8ϩ T cell number in the lymph nodes and spleens of IL-15 KO and IL-15R KO mice compared with their normal counterparts (29, 30). Our observation of a remarkable in- crease in CD8ϩ T cells in IL-2R␣ KO mice provides a reciprocal situation to IL-15 KO mice and IL-15R␣ KO mice. Increase in CD4ϩ T cell numbers was not observed in the lym- FIGURE 10. Roles of Treg cells and Fas played in the immune sys- phoid tissues of IL-2R␣ KO mice except in bone marrow. It is tem biology in IL-2R␣ KO mice: a schematic presentation. In newborn likely that many activated T cells have already infiltrated into var- ϩ Ϫ IL-2R␣ KO mouse, differentiation produces CD4 CD25 T ious inflamed tissues. Therefore, the apparent lack of an increase in ϩ ϩ ϩ Ϫ cells but not CD4 CD25 Treg cells. In the periphery, CD4 CD25 T CD4ϩ T cells cannot be taken as evidence of lacking T cell acti- cells are spontaneously and polyclonally activated and expanded in the vation and expansion. In support of this interpretation, the propor- absence of Treg cells. Some of the autoimmune T cells go to various tion of activated phenotype, as defined by expression of activation tissues/organs and cause inflammation and Fas-based tissue damages. Some autoimmune T cells go to bone marrow where they cause bone marker CD69, increased 2.5- to 4-fold in the lymphoid tissues of IL-2R␣ KO and DM mice in comparison with B6 counterparts marrow depression, leading to inhibition of erythropoiesis and B cell ϩ ϩ lymphopoiesis that contribute, respectively, to anemia and B cell dele- (Fig. 3). This is observed in both CD4 and CD8 T cell popu- tion. We have not yet determined whether the generation of thymocyte lations. It is important to note that in the bone marrow of IL-2R␣ ϩ precursors is inhibited. KO and DM mice, both the total number of CD4 T cells and the The Journal of Immunology 1973

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