Impaired up-regulation of CD25 on CD4؉ T cells in IFN-␥ knockout mice is associated with progression of myocarditis to heart failure

Marina Afanasyeva*†, Dimitrios Georgakopoulos†‡, Diego F. Belardi‡, Djahida Bedja§, DeLisa Fairweather*, Yan Wang*, Ziya Kaya*, Kathleen L. Gabrielson§, E. Rene Rodriguez*, Patrizio Caturegli*, David A. Kass‡, and Noel R. Rose*¶ʈ

Departments of *Pathology, ‡Medicine, and §Comparative Medicine and the ¶W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, MD 21205

Communicated by John W. Littlefield, Johns Hopkins University School of Medicine, Baltimore, MD, November 5, 2004 (received for review March 8, 2004) Inflammation has been recognized increasingly as a critical patho- damage and adverse cardiac remodeling, promoting heart fail- logic component of a number of heart diseases. A mouse model of ure. At the same time, IFN-␥ has been reported to exert direct autoimmune myocarditis was developed to study the role of negative inotropic effects on cardiomyocytes, possibly through immune mediators in the development of cardiac dysfunction. We activation of inducible nitric oxide synthase (13, 14). Based on have found previously that IFN-␥ deficiency promotes inflamma- the latter observation, the prediction can be made that IFN-␥ tion in murine myocarditis. It has been unclear, however, how deficiency is beneficial in a setting of cardiac inflammation. IFN-␥ deficiency in myocarditis affects cardiac function and what Furthermore, it is important to know how IFN-␥ deficiency underlying immune mechanisms are responsible for these effects. affects the composition of the inflammatory myocardial infil- In this work, we show that IFN-␥ knockout (KO) mice have more trate and to identify the underlying immune mechanisms for pronounced systolic and diastolic dysfunction and greater fre- these changes, because the quality as well as the quantity of the quency of progression to dilated cardiomyopathy and heart failure local inflammatory response can determine in vivo organ dam- compared with WT mice. Cardiac dysfunction in the KO mice is age. To address these questions, we studied temporal progression associated with the expansion of activated (CD44high) CD3؉ T cells ؉ ؉ ؉ of cardiac dysfunction as well as associated systemic and local due to reduced of CD4 , but not CD8 , T cells. CD4 T cardiac immune phenomena in IFN-␥ KO mice as compared cells in the KO mice show impaired up-regulation of CD25 upon with WT mice. activation, resulting in the expansion of CD4؉CD44؉CD25؊ T cells and their infiltration into the heart. CD4؉CD25؊ T cells are less Methods ؉ apoptosis-prone compared with the CD25 population, and their Mice. EAM was induced in 10- to 12-week-old female WT and infiltration into the heart is associated with greater severity of IFN-␥ KO BALB͞c mice obtained from The Jackson Labora- ␥ myocarditis. We conclude that IFN- deficiency in autoimmune tory and maintained in The Johns Hopkins University School of myocarditis is associated with preferential expansion of ؉ ؉ ؊ Medicine conventional animal facility. The animal work was CD4 CD44 CD25 T cells resulting in increased cardiac inflamma- approved by the Animal Care and Use Committee of The Johns tion. An exaggerated inflammatory response in IFN-␥ KO mice Hopkins University. causes cardiac dysfunction, leading to dilated cardiomyopathy and heart failure. Induction and Assessment of EAM. CM immunization and histologic assessment were performed as described in ref. 11. Gross scores activation-induced cell death ͉ autoimmunity ͉ pressure–volume relations were based on the extent of white discoloration on a scale from 0 to 5 as described for histologic scores. nflammation and autoimmunity increasingly have been rec- Iognized as important pathogenic components in cardiac dis- Assessment of Cardiac Function. Pressure–volume studies were eases and the development of cardiac dysfunction. A number of performed as described in ref. 15. The time constant of isovolu- ␣ cytokines, including IL-6 and tumor necrosis factor , have been mic relaxation (␶) was derived by using an assumption of shown to contribute to the progression of heart failure. In monoexponential pressure decay with a nonzero asymptote (16). inflammatory heart diseases, cytokines can affect disease out- Transthoracic echocardiography was performed as described in comes by regulating the degree of inflammation, by mediating refs. 17 and 18. cardiac remodeling through their effects on fibrosis and cardi- omyocyte hypertrophy, or by directly influencing cardiomyocyte In Vitro Stimulation. Spleen and peripheral blood samples were function. ␥ treated for 5 min with ACK lysing buffer (pH 7.4) (BioWhit- IFN- , a prototypic T helper 1 cytokine, mainly produced by taker). Live cells were counted by trypan blue exclusion. Cells natural killer and T cells, stimulates T helper 1 development, were cultured in 24-well plates at an initial cell density of 5 ϫ 107 activates macrophages, induces MHC class II expression, promotes cells per ml in complete RPMI medium 1640 (Life Technologies, delayed-type hypersensitivity reactions, plays an important role in Grand Island, NY) with the additional supplementation de- clearing intracellular bacteria and parasites, and exhibits antiviral scribed in ref. 19, in the presence of 10 ␮g͞ml anti-CD3 mAb or activity (1). Despite its reported proinflammatory activity (2–4), 10 ␮g͞ml anti-CD3 and 5 ␮g͞ml anti-CD28 for 12 h or in the IFN-␥ has been shown to suppress inflammation in many animal models of autoimmune disease (5–7). It has been demonstrated ␥ previously that IFN- suppresses acute inflammation and that Abbreviations: CM, cardiac myosin; EAM, experimental autoimmune myocarditis; KO, IFN-␥-deficient mice develop an exaggerated inflammatory re- knockout; DCM, dilated cardiomyopathy; 7AAD, 7-aminoactinomycin D. sponse in a model of cardiac myosin (CM)-induced experimental †M.A. and D.G. contributed equally to this work. autoimmune myocarditis (EAM) (8–12). ʈTo whom correspondence should be addressed at: Department of Pathology, Johns It has been unclear, however, how IFN-␥ deficiency in EAM Hopkins University School of Medicine, 720 Rutland Avenue, Ross Building, Room 659, affects cardiac function. Exaggerated acute myocardial inflam- Baltimore, MD 21205. E-mail: [email protected]. mation in IFN-␥ knockout (KO) mice may lead to cardiomyocyte © 2004 by The National Academy of Sciences of the USA

180–185 ͉ PNAS ͉ January 4, 2005 ͉ vol. 102 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408241102 Downloaded by guest on September 23, 2021 Fig. 1. IFN-␥ deficiency in EAM is associated with greater cardiac dysfunction. Shown are heart-weight͞body-weight ratio (HW͞BW) (a); cardiac output (CO) (b); maximal rate of pressure development (dP͞dtmax)(c); stroke work-end-diastolic volume relations, or preload-recruitable stroke work (PRSW) (d); time constant of isovolumic pressure decay (␶)(e); and end-diastolic elastance (Eed) normalized to heart weight, which represents passive stiffness (f)inWT(F) and IFN-␥ KO (E) mice. Day 0 represents unimmunized age-matched controls. Data in b–f were obtained by using the in vivo pressure–volume method. Data are presented as mean Ϯ SE. Day 0, n ϭ 6 (WT) and n ϭ 4 (KO); days 21–24, n ϭ 9 in both groups; days 31–33, n ϭ 12 in both groups; days 42–44, n ϭ 9 (WT) and n ϭ 8 (KO); days 58–62, n ϭ 6 in both groups. *, P Ͻ 0.05, compared with WT mice during the same time point.

presence of 10 ␮g͞ml CM for 3 days. Peripheral blood cells were mation declined and did not differ significantly between the two stimulated for4hwith50ng͞ml phorbol 12-myristate 13-acetate groups. (Life Technologies) and 1 ␮mol͞liter ionomycin (Sigma). Effects of IFN-␥ Deficiency on Cardiac Function. Increased myocar- Flow Cytometry. Mouse hearts were digested, and flow cytometry ditis scores in IFN-␥ KO mice were accompanied by increases in was performed as described in refs. 20 and 21. CD25ϩ cells were heart weight (data not shown) and the heart-weight͞body-weight detected with allophycocyanin-labeled Ab (clone PC-61, Pharm- ratio indicative of the development of cardiomyopathy (Fig. 1a). ingen). Cells were fixed overnight with Cytofix͞Cytoperm buffer To study how IFN-␥ deficiency affects cardiac function in EAM, (pH 7.4) (Pharmingen). Annexin V and 7-aminoactinomycin D we performed in vivo measurements of left-ventricular function. (7AAD) stains and assessment of heart cells were performed on We found greater impairment of systolic and diastolic function unfixed cells. in the KO compared with the WT mice (Fig. 1 b–f). This impairment was most pronounced during the acute phase of Statistical Analyses. The Mann–Whitney U test was used to disease (days 21–24) with indices of cardiac function improving compare myocarditis scores, and Student’s t test was used for by days 42–44. Day 58–62 measurements, however, were indic- other comparisons. Associations between cellular populations in ative of further decompensation in the KO mice associated with the heart were assessed using linear regression. The data were the second peak of increase in heart-weight͞body-weight ratios. analyzed by using SIGMASTAT 3.01 software (SPSS, Chicago). P Յ Impaired cardiac performance, including such parameters as 0.05 was considered statistically significant. cardiac output, maximal rate of pressure development (dP͞ dtmax), time constant of pressure decay (␶), in the KO mice highly

Results correlated with cardiac inflammation as assessed by histologic MEDICAL SCIENCES Increased Cardiac Pathology in IFN-␥ KO Mice. We found that the KO scores (data not shown); such an association was especially mice had significantly higher gross and histologic scores of pronounced during the acute phase of disease. disease not only during the acute phase of disease (days 21–24) The KO mice had higher frequency of progression to dilated but also at later time points, including days 31–33, 42–44, and cardiomyopathy (DCM) (12 of 35 in KO vs. 1 of 33 in WT) and 58–62 (chronic phase) after immunization (Fig. 5 a and b, which heart failure (13 of 39 in KO vs. 1 of 33 in WT) (Fig. 2 a–c). Mice is published as supporting information on the PNAS web site). with DCM had large end-diastolic volumes (Ն50 ␮l) and signif- On microscopic examination, the KO mice exhibited more icantly reduced systolic performance, as assessed by cardiac intense inflammatory infiltration, especially during the acute output, ejection fraction, dP͞dtmax, stroke work, and maximal phase, and greater cardiac fibrosis, which was more pronounced power as well as impaired diastolic relaxation (␶) and gross at later time points. Flow-cytometric examination of the heart cardiac enlargement with dilatation of all four chambers (Fig. 2 infiltrates in the WT mice revealed that inflammation (assessed d–h). The higher frequency of DCM in IFN-␥ KO mice also was by percentage of infiltrating CD45ϩ leukocytes) peaked by day demonstrated by echocardiography, which was used to serially 15 (Fig. 5c). In contrast, peak inflammation in the KO mice monitor disease progression in the same animal (Fig. 2 i and j). occurred on days 21–24 and, at this time point, was associated Mice with heart failure had markedly reduced indices of systolic with significantly higher percentages of infiltrating leukocytes function associated with reduced body weight, ascites, and a compared with the WT mice. At later times, myocardial inflam- typical lung pathology that included pulmonary congestion with

Afanasyeva et al. PNAS ͉ January 4, 2005 ͉ vol. 102 ͉ no. 1 ͉ 181 Downloaded by guest on September 23, 2021 Fig. 2. Increased frequency of progression to DCM in IFN-␥ KO mice in EAM. Pressure–volume loops obtained at steady state and during the inferior vena cava occlusion (a–c) and heart cross sections (d–f) from a representative unimmunized KO mouse (a and d), an immunized WT mouse (day 23) (b and e), and an immunized KO mouse (day 23) (c and f). In c, note the increased left-ventricular volumes, reduced stroke volume, reduced systolic pressures, and increased diastolic passive stiffness (the slope of the diastolic curve), characteristic of DCM. (d–f) Hematoxylin͞eosin stain. (Original magnification: ϫ4.) (g and h) Representative gross hearts from WT (g) and KO (h) mouse during acute EAM. (i and j) M-mode echocardiograms from the same IFN-␥ KO mouse on days 19 (i) and 29 (j) after immunization. (k) Normal lung section from WT with EAM. (Original magnification: ϫ200.) (l) Lung section from a KO mouse with heart failure, exhibiting congestion, secondary pneumonia, and hemosiderin-laden macrophages (arrows, heart failure cells). (Original magnification: ϫ80.) secondary pneumonia and ‘‘heart failure cells’’ (Fig. 2 k and l). CD4؉ vs. CD8؉ T Cell Subsets in EAM in IFN-␥ Deficiency. It has been DCM was observed starting day 23 after immunization, and its shown previously that IFN-␥ controls the expansion of both severity, as assessed by end-diastolic volume, significantly cor- CD4ϩ and CD8ϩ T cells in response to immune activation related with the degree of myocardial fibrosis (data not shown). (22–24). We reported previously that IFN-␥ deficiency in EAM leads to expansion of both subsets in the spleen (11). It has been IFN-␥ Deficiency Leads to Reduced Apoptosis and Systemic Expansion unclear, however, which subset is more important in promoting of Activated T Cells. To assess the immune mechanisms of en- disease in IFN-␥ KO mice. To investigate the role of CD4ϩ vs. hanced disease in IFN-␥ KO mice, we first studied the peripheral CD8ϩ T cells, we assessed the degree of apoptosis in both subsets blood T cell population after CM immunization. The KO mice in lymph nodes and spleen. CD4ϩ, but not CD8ϩ, T cells in had significantly greater percentages of CD3ϩ T cells on day 16 IFN-␥ KO mice consistently showed less apoptosis compared after immunization (see Table 1, which is published as support- with T cells from WT mice (Fig. 3 c and d). To study a more ing information on the PNAS web site). Percentages of activated disease-relevant population of T cells, we used flow cytometry CD3ϩ T cells expressing high levels of CD44 (CD44high) were on heart-derived cells that were enriched for leukocytes through increased significantly in the KO mice starting on day 3 after depletion of cardiomyocytes (21). We found that CD4ϩ T cells immunization with the absolute difference between the two predominated over CD8ϩ T cells in the heart infiltrate in both groups being the greatest on day 16 (Table 1). Interestingly, in genotypes at all time points after immunization (data not the KO mice this population of activated T cells expanded up to shown). During the acute phase, there was an increased influx of day 16, whereas in the WT mice these cells stopped expanding both CD4ϩ and CD8ϩ T cells in the heart in IFN-␥ KO mice around day 9. To test the hypothesis that the greater expansion compared with WT mice (Fig. 3 e and f). However, only the of activated T cells in the KO mice is associated with reduced proportion of CD4ϩ, but not CD8ϩ, T cells of total infiltrating activation-induced cell death, we assessed the number of T cells leukocytes was significantly increased in IFN-␥ KO mice (Fig. 3 undergoing early apoptosis (annexin Vϩ7AADϪ). We found that g and h). During the acute phase of EAM, IFN-␥ KO mice had the KO mice had a significantly lower percentage of CD3ϩ T significantly increased CD4͞CD8 ratios in the myocardium cells undergoing early apoptosis on day 9 after immunization compared with WT mice (5.2 Ϯ 3.3 in KO vs. 1.7 Ϯ 0.9 in WT, (Table 1) than the WT mice. Consistent with the observations in P ϭ 0.004). peripheral blood, IFN-␥ KO mice had increased proportions of CD3ϩ T cells in the acute heart infiltrate (data not shown), and IFN-␥ KO Mice Have Decreased Expression of CD25 on CD4؉ T Cells in these T cells had greater intensity of CD44 expression (Fig. 3 a EAM. To further characterize the pathologically relevant subset of and b), indicating the presence of not only quantitative but also CD4ϩ T cells, we looked at the expression of CD25, the qualitative differences in the heart infiltrate compared with the ␣-subunit of the IL-2 receptor, on their surface. Expression of WT mice. CD25 on CD4ϩ T cells trafficking through normal hearts of

182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408241102 Afanasyeva et al. Downloaded by guest on September 23, 2021 Fig. 3. Expansion of CD4ϩ T cells in IFN-␥ KO mice. (a) Percentage of CD44high cells of CD3ϩ T cells in the hearts of WT (F) and IFN-␥ KO (E) mice on days 21–24 after immunization. (b) Mean fluorescence intensity (MFI) of CD44 staining on CD3ϩ T cells in the hearts of WT (F) and KO (E) mice on days 21–24 after immunization. (c and d) Percentage of CD4ϩ (c) and CD8ϩ (d) T cells undergoing early apoptosis (annexin Vϩ7AADϪ)inWT(F) and KO (E) mice in lymph nodes and spleen day 16 after immunization. (e and f) Representative flow-cytometry plots of CD45ϩCD4ϩ (e) and CD45ϩ CD8ϩ (f) T cells (upper right quadrants) from WT and KO. Numbers represent percentages of either CD4ϩ or CD8ϩ T cells of total heart cells. (g and h) Percentage of CD8ϩ (g) and CD4ϩ (h) T cells of infiltrating CD45ϩ leukocytes in WT (F) and KO (E) mice in the heart on days 21–24 after immunization. Each symbol represents an individual mouse.

unimmunized mice was similar between the KO and WT (Fig. regulation of CD25 on CD4ϩ T cells in both groups, but the 4a). The acute phase of EAM, however, was associated with a percentage of CD25high cells of total CD4ϩ T cells was consis- decreased proportion of CD4ϩCD25ϩ T cells in the heart in tently lower in the KO mice (Fig. 4f). This activation also resulted IFN-␥ KO mice compared with either immunized WT or in a significantly greater percentage of CD4ϩCD44high T cells in unimmunized KO mice (Fig. 4a). Importantly, more severe IFN-␥ KO mice compared with WT mice (data not shown). To myocardial inflammation, as assessed by percentages of CD45ϩ gain greater insight into how the impaired up-regulation of CD25 leukocytes in the heart, correlated with reduced proportions of on CD4ϩ T cells might be related to reduced apoptosis in IFN-␥ CD4ϩCD25ϩ T cells infiltrating the heart (Fig. 4b). There was no KO mice, we looked at the degree of apoptosis in CD25ϩ and difference in CD25 expression on CD4ϩ T cells in the peripheral CD25Ϫ populations of CD4ϩ T cells from peripheral blood, blood between the two genotypes at baseline (data not shown) lymph nodes, and spleen of immunized mice. We found that or on days 3 and 16 after immunization. IFN-␥ KO mice, CD4ϩCD25ϩ T cells were consistently more death-prone than MEDICAL SCIENCES however, had a significantly smaller percentage of CD4ϩCD25ϩ the CD4ϩCD25Ϫ T cell population regardless of the genotype T cells in the peripheral blood on day 9 after immunization (Fig. (Fig. 4g). Finally, we found that CD4ϩ T cells collected from 4c) compared with WT mice. This time point was associated with immunized mice and treated with anti-CD3 and anti-CD28 an increase in CD25 expression compared with the baseline in mAbs that expressed high levels of CD25 had higher percentages WT mice, but this up-regulation was not observed in KO mice. of dead cells compared with those with intermediate levels of In EAM, day 9 generally corresponded to the peak activation of CD25 (Fig. 4 h and i). in the periphery in response to the second injection of CM on day 7. Discussion These findings led us to postulate that IFN-␥ deficiency in Our results demonstrate that IFN-␥ deficiency intensifies disease EAM is associated with the impairment of activation-induced in EAM, producing an enhanced proinflammatory response that up-regulation of CD25 on CD4ϩ T cells. This hypothesis was leads to greater cardiac damage and dysfunction. Increased supported by our observation that in vitro stimulation with either myocardial inflammation in the absence of IFN-␥ was associated 12-myristate 13-acetate plus ionomycin or CM resulted in a with reduced apoptosis and expansion of activated T cells. The lower CD25 expression on CD4ϩ T cells in IFN-␥ KO mice (Fig. reduction in apoptosis was characteristic of the CD4ϩ, but not 4 d and e). In vitro stimulation of splenocytes with either the CD8ϩ, T cell subset, and CD4ϩ T cells were predominant in anti-CD3 or anti-CD3 and anti-CD28 mAbs resulted in up- the enhanced myocardial lesions. The importance of CD4ϩ T

Afanasyeva et al. PNAS ͉ January 4, 2005 ͉ vol. 102 ͉ no. 1 ͉ 183 Downloaded by guest on September 23, 2021 Fig. 4. IFN-␥ deficiency is associated with reduced expression of CD25 on CD4ϩ T cells in EAM. (a) Percentages of CD4ϩ T cells expressing CD25 in the hearts of unimmunized (day 0) and immunized (days 22–24) WT (F) and KO (E) mice. (b) Association between percentages of CD25ϩ of CD4ϩ T cells and percentages of CD45ϩ leukocytes in the heart in WT (F) and KO (E) mice on days 22–24 after immunization. (c) Percentages of CD25ϩ of CD4ϩ T cells in the peripheral blood in WT (F) and KO (E) mice. The data are representative of two experiments. (d and e) Representative histogram showing CD25 expression on CD4ϩ T cells from either peripheral blood (stimulated with 12-myristate 13-acetate and ionomycin) (d) or spleen and lymph nodes (stimulated with CM) (e) day 21 after immunization. (f) Percentages of CD25high of CD4ϩ T cells from spleens of WT (F) and KO (E) mice (stimulated with either anti-CD3 or anti-CD3 and anti-CD28 mAbs) day 16 after immunization. (g) Percentages of live (7AADϪannexin VϪ) CD25Ϫ or CD25ϩ CD4ϩ T cells from peripheral blood, lymph nodes, and spleen of WT (F) and KO (E) mice day 16 after immunization. All comparisons between CD25ϩ and CD25Ϫ cells in each organ and in both WT and KO were statistically significant. (h and i) Cells were collected from lymph nodes (h) or spleen (i) in WT (filled symbols) and KO (open symbols) mice on day 16 after immunization and stimulated with anti-CD3 and anti-CD28 mAbs. Percentages of live lymph node-derived CD4ϩ T cells expressing intermediate levels of CD25 (CD25interm) versus those expressing high levels of CD25 (CD25high). The lines connect values from the same mouse. Each symbol represents an individual mouse.

cells in WT mice in EAM has been demonstrated by Smith and mice are not different from those in WT mice. Similarly, there Allen (25) in experiments involving CD4ϩ T cell depletion and is no difference in CD25 expression on between the adoptive transfer. Importantly, we have found that percentages two groups (data not shown), which indicates that IFN-␥ defi- of CD4ϩ T cells, but not the total numbers of infiltrating ciency is not associated with the lack of thymic production of leukocytes, in the heart infiltrate in WT mice significantly CD4ϩCD25ϩ regulatory T cells. Accordingly, IFN-␥ KO mice do correlated with the degree of systolic dysfunction and progres- not spontaneously develop autoimmune diseases. sion to DCM during the chronic phase of EAM (21). In this IFN-␥ KO mice exhibit enhanced autoimmune response when work, we demonstrate that increased percentages in CD4ϩ T autoreactive lymphocytes are exogenously stimulated in the cells in the heart are associated with greater cardiac dysfunction presence of a strong adjuvant. The difference in CD25 expres- in IFN-␥ KO mice. sion was observed only upon immunization-induced activation of To our knowledge, this study is the first to demonstrate that T cells and was pronounced in the peripheral blood 9 days after IFN-␥ deficiency in autoimmune disease is associated with immunization, a time point that corresponds to the peak acti- reduced proportions of CD4ϩ T cells expressing CD25. vation of peripheral lymphocytes in EAM. Significant differ- CD4ϩCD25ϩ T cells have been shown to suppress autoaggressive ences in percentages of CD4ϩCD25ϩ T cells in the heart were T cell clones and, therefore, are critical in mediating peripheral observed only during the acute phase of EAM but not before tolerance (26, 27). Mice lacking CD25 develop lymphoprolif- immunization or during the recovery phase. Our in vitro exper- eration and spontaneous autoimmune diseases, and human iments demonstrated that upon activation, CD4ϩ T cells from CD25 deficiency is associated with the occurrence of autoreac- IFN-␥ KO mice up-regulated CD25 to a significantly smaller tive T cells (28, 29). Classic regulatory CD4ϩCD25ϩ T cells degree than those from the WT mice. Thus, the difference in develop in the thymus and, in BALB͞c mice, represent Ϸ6% of CD25 expression cannot be explained by reduced thymic pro- peripheral blood CD4ϩ T cells (30, 31). We have found that the duction of classic regulatory T cells in IFN-␥ KO mice but, levels of CD25 expression on CD4ϩ T cells in naı¨ve IFN-␥ KO rather, by the impaired up-regulation of CD25 upon activation.

184 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408241102 Afanasyeva et al. Downloaded by guest on September 23, 2021 It has been reported that the CD4ϩCD25ϩ regulatory T cells are regulation in autoimmune myocarditis is manifested by the characterized by the high expression of CD62L, whereas resulting expansion of the autoaggressive CD4ϩCD25Ϫ T cells. CD4ϩCD25ϩ activated T cells have low expression of CD62L We also have found increased expression of CD44 on T cells (32, 33). We found that the predominant majority of in IFN-␥ KO mice both in the periphery and the heart, indicative CD4ϩCD25ϩ T cells in EAM are CD62Llow (data not shown), of a higher proportion of -experienced population (37). further supporting the notion that these T cells represent an Similarly, Chu et al. (22) demonstrated the expansion of ϩ activated population. CD44highCD4 T cells in IFN-␥ KO in a model of experimental Our findings suggest that IFN-␥ deficiency is associated with autoimmune encephalomyelitis. The disproportionate expansion high the disturbed kinetics of up-regulation of CD25 during CD4ϩ T of CD44 T cells is likely the result of impaired activation- ␥ cell activation. The abnormalities of T cell activation in the induced cell death in the absence of IFN- , which targets absence of IFN-␥ are associated with reduced susceptibility to activated, but not naı¨ve, T cells. Myocardial infiltration by the aggressive CD4ϩ T cells in activation-induced cell death, which is critical for physiologic ␥ termination of T cell responses (34). We demonstrate that CD4ϩ IFN- KO mice results in increased cardiac damage, remodeling, T cells from IFN-␥ KO mice are less susceptible to apoptosis, dysfunction, and eventually heart failure. Cardiac damage due to which accords with observations in other animal models (35). inflammation leads to replacement fibrosis, which is associated with the development of DCM in the KO mice. The latter IL-2 receptor signaling has been shown to be involved in inducing observation agrees with studies in humans demonstrating that not only prosurvival but also proapoptosis pathways and is hearts from DCM patients have increased collagen content (38). critical for activation-induced cell death in CD4ϩ T cells (36). These findings provide insights into the immune mechanisms CD25 expression leads to increased affinity of IL-2 receptor and, leading to the expansion of autoaggressive T cell populations in therefore, might contribute to the shift from prosurvival to the context of IFN-␥ deficiency. Further studies are needed to proapoptotic signaling (36). In this regard, we found that delineate the exact mechanisms by which the enhanced inflam- CD4ϩCD25ϩ T cells are more apoptosis-prone than ϩ Ϫ mation in the myocardium causes cardiomyocyte damage and CD4 CD25 T cells. The kinetics as well as the degree of dysfunction leading to heart failure. Our findings not only may activation-induced expression of CD25 may determine to which be relevant to the immune involvement in the development of extent the effector CD4ϩ T cells expand or contract. Thus, the ϩ cardiomyopathy but also may be applicable to a broad range of impaired up-regulation of CD25 on CD4 T cells in IFN-␥ autoimmune diseases. deficiency may be linked to the inability to physiologically terminate the expansion of the effector T cells and, therefore, to This work was supported by National Institutes of Health Grants promote autoimmune responses. The importance of such dys- HL67290, HL70729, and AI51835.

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Afanasyeva et al. PNAS ͉ January 4, 2005 ͉ vol. 102 ͉ no. 1 ͉ 185 Downloaded by guest on September 23, 2021