Dendritic Cell KLF2 Expression Regulates Activation and Proatherogenic Immune Responses

This information is current as Noah Alberts-Grill, Daniel Engelbertsen, Dexiu Bu, Amanda of September 28, 2021. Foks, Nir Grabie, Jan M. Herter, Felicia Kuperwaser, Tao Chen, Gina Destefano, Petr Jarolim and Andrew H. Lichtman J Immunol 2016; 197:4651-4662; Prepublished online 11

November 2016; Downloaded from doi: 10.4049/jimmunol.1600206 http://www.jimmunol.org/content/197/12/4651

Supplementary http://www.jimmunol.org/content/suppl/2016/11/11/jimmunol.160020 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 59 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/197/12/4651.full#ref-list-1

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Dendritic Cell KLF2 Expression Regulates T Cell Activation and Proatherogenic Immune Responses

Noah Alberts-Grill,1 Daniel Engelbertsen,1 Dexiu Bu, Amanda Foks, Nir Grabie, Jan M. Herter, Felicia Kuperwaser, Tao Chen, Gina Destefano, Petr Jarolim, and Andrew H. Lichtman

Dendritic cells (DCs) have been implicated as important regulators of innate and adaptive inflammation in many diseases, including . However, the molecular mechanisms by which DCs mitigate or promote inflammatory pathogenesis are only par- tially understood. Previous studies have shown an important anti-inflammatory role for the Kruppel-like€ factor 2 (KLF2) in regulating activation of various cell types that participate in atherosclerotic lesion development, including endothelial cells, macrophages, and T cells. We used a pan-DC, CD11c-specific cre-lox knockout mouse model to assess the role of KLF2 in DC activation, function, and control of inflammation in the context of hypercholesterolemia and atherosclerosis. We found that Downloaded from KLF2 deficiency enhanced surface expression of costimulatory molecules CD40 and CD86 in DCs and promoted increased T cell proliferation and . Transplant of bone marrow from mice with KLF2-deficient DCs into Ldlr2/2 mice aggravated atherosclerosis compared with control mice, most likely due to heightened vascular inflammation evidenced by increased DC presence within lesions, enhanced T cell activation and cytokine production, and increased cell death in atherosclerotic lesions. Taken together, these data indicate that KLF2 governs the degree of DC activation and hence the intensity of proatherogenic T cell responses. The Journal of Immunology, 2016, 197: 4651–4662. http://www.jimmunol.org/

therosclerosis is a chronic inflammatory disease involv- have also shown that the maintenance of atherosclerosis-suppressing ing both the innate and adaptive arms of the immune regulatory T cell (Treg) responses requires MyD88-dependent sig- A response that is characterized by the development of naling in classical, Flt3-Flt3L–dependent DCs, the loss of which lipid-laden plaques in the arterial wall (1–4). Dendritic cells (DCs), aggravates atherosclerosis (10, 11). Thus, DCs are involved in the a class of innate immune cells that function at the junction of complex interplay of proinflammatory and regulatory mechanisms innate and adaptive immunity, have been shown to play a central that impact atherosclerosis.

role in the initiation of atherosclerosis. During early lesion de- Whereas the generation of DCs with proinflammatory or tol- by guest on September 28, 2021 velopment, DCs as well as macrophages accumulate and trans- erogenic T cell priming capabilities in vitro has been well estab- form into the early foam cells of the so-called “fatty streak” (5). lished (9, 12–14), the molecular mechanisms by which DCs DCs are also responsible for priming proatherogenic T cell re- mature and function in vivo to drive either pro- or antiatherogenic sponses against modified lipid and other atherosclerosis-related T cell responses in the context of hypercholesterolemia are poorly Ags (6, 7). However, DCs have also been shown to play a pro- understood. Kruppel-like€ factor 2 (KLF2) is a transcription factor tective role in atherosclerosis. DC-mediated nasal immunization with well-established regulatory functions, including maintenance with heat shock 65 ameliorated atherosclerosis in hyper- of quiescence in numerous cell types important in atherosclerosis, cholesterolemic mice (8), as did the adoptive transfer of tolero- such as endothelial cells, macrophages, and T cells (15–21). Re- genic DCs loaded with apolipoprotein B-100 (9). Other studies cent research has shown that KLF2 modulates development and inflammatory activity in macrophages and neutrophils (17, 22, 23). hi Department of , Brigham and Women’s Hospital, Harvard Medical School, KLF2 hemizygous mice showed increased inflammatory Ly-6C Boston, MA 02460 monocytes in the circulation and increased recruitment of Ly-6Chi 1N.A.-G. and D.E. are joint first authors. macrophages to the peritoneum (23). Importantly, pan-myeloid ORCIDs: 0000-0003-1982-212X (D.B.); 0000-0002-0613-2640 (T.C.); 0000-0002- of KLF2 in Lyz2cre Klf2fl/fl mice led to spontaneous mac- 4159-279X (G.D.); 0000-0002-9612-8652 (P.J.). rophage activation and a fatal sepsis-like innate immune response Received for publication February 4, 2016. Accepted for publication October 17, against bacterial infection (17). 2016. With regard to T cell responses, studies in our laboratory have This work was supported by National Institutes of Health Grants R01 HL087282 and RO1 HL121363 (to A.H.L.) and T32 HL07627 (to N.A.-G.). D.E. was supported by shown that statin-induced expression of KLF2 negatively regulates fellowships from the Swedish Heart- Foundation and The Tegger Foundation. inflammatory functions of T cells (18). DCs, the principal APCs Address correspondence and reprint requests to Dr. Andrew H. Lichtman, Depart- for naive T cells, express relatively low levels of KLF2 mRNA, ment of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 77 and the biological significance of DC-KLF2 is not clear. There- Avenue Louis Pasteur, Boston, MA 02115. E-mail address: [email protected] fore, we examined the effects of Klf2 deletion in CD11c- The online version of this article contains supplemental material. expressing cells on DC and function, and on T cell Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AICD, activation- induced cell death; BMDC, bone marrow–derived DC; BMDM, bone marrow– priming and activation in vitro and in vivo. We also determined the derived macrophage; DC, dendritic cell; KLF2, Kruppel-like€ factor 2; KO, knockout; effects of DC-KLF2 deficiency on atherosclerosis in mice by LDL, low-density lipoprotein; qRT-PCR, quantitative RT-PCR; Treg, regulatory transplanting bone marrow from mice with KLF2-deficient DCs T cell. into low-density lipoprotein (LDL) –deficient (Ldlr2/2) Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 mice and characterizing lesion development after feeding them a www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600206 4652 DC-KLF2 AND THE PROATHEROGENIC IMMUNE RESPONSE high-fat, high-cholesterol diet for 10 wk. We found that loss of DC- Flow cytometry KLF2 exacerbated atherosclerosis while paradoxically inducing BMDCs were stained using the following Abs (BioLegend): CD11c (N418), profound leukopenia, primarily via loss of T cells. KLF2-deficient I-Ab (AF6-120.1), CD40 (3/23), and CD86 (GL-1). BMDC viability was DCs expressed higher levels of costimulatory molecules such as assessed using Zombie Aqua fixable viability dye (BioLegend). Staining CD40 and CD86 following LPS-induced maturation, and they were for monocyte and DC populations in peripheral blood leukocyte prepara- more efficient at stimulating both CD4+ and CD8+ T cell prolifer- tions was done using Abs against CD11c (N418), CD11b (M1/70), Ly-6C (HK1.4), I-A/I-E (M5/114.15.2), Flt3 (A2F10), CD90.2 (53-2.1), B220 ation, cytokine production, and apoptosis. (RA3-6B2), Ly-6G (1AB), NK1.1 (PK136), and CD49b (DX5, Supplemental Fig. 1). Splenocytes and peritoneal macrophages were stained for mac- rophage markers CD11b (M1/70), F4/80 (BM8), and CD11c (N418). Materials and Methods T cell staining of splenocytes was done using the following Abs: CD3 Mice (145-2C11), CD4 (RM4-5), CD8 (53-6.7), and B220 (RA3-6B2). Spleno- + + All animals used in this study were bred and housed in the pathogen-free cytes were separately stained for activated (CD25 )CD4 T cells and Tregs (CD25+Foxp3+) using the following Abs in addition to Zombie Aqua fixable facility at the Warren Alpert Building (Harvard Medical School, Boston, viability dye (BioLegend): CD4 (RM4-5), CD25 (PC61), and Foxp3 (FJK- MA) in accordance with Institutional Animal Care and Use Committee guidelines. LDL receptor knockout (KO) mice (B6.129S7-Ldlrtm1Her) and 16S). Permeabilization of cells for Foxp3 staining was done using a Foxp3 CD11c-cre mice [C57BL/6J-Tg(Itgax-cre,-EGFP)4097Ach/J] were pur- staining buffer set (eBioscience). Measurements of T cell apoptosis in lymph chased from The Jackson Laboratory (Bar Harbor, ME). Klf2fl/fl mice were node cells from OVA-immunized mice were done by staining with CD90.2 a gift from Kahn and coworkers (24). Klf2fl/fl mice were bred with Itgaxcre- (53-2.1), CD4 (RM4-5), and CD8 (53-6.7) mAbs (BioLegend) and a PE– cre mice to yield Itgaxcre-Klf2fl/fl mice, which have conditional deficiency annexin V apoptosis detection kit I (BD Biosciences). Cell viability was fl/fl assessed using 7-aminoactinomycin D (7-AAD) viability dye (BD Biosci- of KLF2 expression in CD11c-expressing cells, including most DCs. Klf2

ences). Apoptotic cell percentages were calculated by adding early (annexin Downloaded from littermates lacking cre expression were used as control animals. V+) and late (annexin V+7-AAD+) events. For proliferation studies, lymph Cells and treatment node cells were loaded with CFSE prior to culture as previously noted and stained with CD90.2 (53-2.1) and CD4 (RM4-5) mAbs and 7-AAD via- Bone marrow–derived DCs (BMDCs) or bone marrow–derived macro- bility dye (BD Biosciences). All samples were acquired on a DxP11 flow phages (BMDMs) were generated as previously described (25, 26). In cytometer (Cytek). Flow cytometry data were analyzed using FlowJo v10 short, bone marrow cells were cultured in complete RPMI 1640 medium analysis software (Tree Star). supplemented with L-glutamine, sodium pyruvate, MEM–nonessential amino acids, and either 20 ng/ml GM-CSF (BMDCs; PeproTech) or 10 ng/ml Immunohistochemistry and immunofluorescence staining of http://www.jimmunol.org/ M-CSF (BMDMs; PeproTech) for 5–7 d, and BMDCs/BMDMs were har- aortic lesions vested from cultures. For in vitro experiments, BMDCs were matured by treatment with LPS (1 mg/ml) (Sigma-Aldrich) for 24 h before use. For Frozen sections (7 mm) of aortic sinus lesions were stained with Abs measurements of DC Klf2 expression, some BMDC preparations were pre- specific for CD4 (RM4-5; BD Pharmingen) macrophage marker Mac-3 treated for 24 h with low-dose simvastatin (0.5 mM) and rapamycin (1 nM) (M3/84; BD Pharmingen) and neutrophil marker recognizing Ly-6C and prior to LPS activation. Ly-6G (NIMP-R14; Abcam) and visualized with appropriate secondary Abs. Neutral lipid accumulation in lesion sections was measured by Oil Bone marrow transplant Red O staining. Nuclei for all immunohistochemistry stains were coun- terstained using Gil’s hematoxylin. Immunohistochemistry stains were 2/2 Male and female 8-wk-old Ldlr mice were subjected to 950 rad of total visualized with a Nikon Microphot-Fxa microscope (Nikon) equipped with body irradiation delivered 4 h apart in two doses (450 and 500 rad) and an FX-35-DX digital camera (Nikon). Images were captured using either by guest on September 28, 2021 6 fl/fl reconstituted with 2 3 10 bone marrow cells from Itgaxcre-Klf2 or 34/0.13, 310/0.45, or 340/0.70 objective lens as indicated using the fl/fl Klf2 mice via tail vein injection. Bone marrow recipients received ACT-2U imaging software (Nikon). For immunofluorescence stains, sec- sulfamethoxazole/trimethoprim (Sulfatrim) treatment administered in drinking tions were stained with biotinylated anti-CD11c (HL3) mAb and visual- water for 1 wk prior to and 4 wk following bone marrow transplantation. ized with streptavidin-conjugated DyLight 549 (Jackson ImmunoResearch All animals were allowed to recover on a chow diet for 6 wk after bone Laboratories, West Grove, PA). Sections were mounted in ProLong Gold marrow transplantation and then fed an atherogenic high-fat diet con- antifade mounting medium with DAPI (Thermo Fisher Scientific) and taining 1.25% cholesterol (Research Diets, catalog no. D1218C) (27) for visualized with an Olympus FluoView FV1000 microscope (Olympus) and 10 wk. Olympus DP72 digital camera (Olympus). Images were captured using either a 310/0.30 or 320/0.75 objective lens and the FV10-ASW imaging Histological analysis and morphometric analysis of aortic software (Olympus). All images were modified after capture to enhance atherosclerosis brightness and contrast independently for each color channel using the level function in Adobe Photoshop (Adobe Systems) After sacrifice following 10 wk of atherogenic diet feeding, aortic roots were dissected, embedded in OCT, and serial frozen section sections were Quantitative RT-PCR analyses prepared. Analysis of atherosclerotic lesion size was performed on five Oil Red O–stained cryosections (10 mm each) spanning 160 mm of the three- levels in RNA samples were determined using a StepO- valve area of the aortic root, as described (28, 29). nePlus real-time PCR system (Applied Biosystems). RNA was extracted using an RNeasy mini kit (Qiagen) per the manufacturer’s instructions, and Immunization cDNA was made using a ThermoScript RT-PCR kit (Thermo Fisher Sci- entific). Quantitative RT-PCR (qRT-PCR) was performed with forward and Itgaxcre-Klf2fl/fl For OVA immunization/T cell restimulation studies, or reverse primer sets as follows: b-actin forward, 59-TCC TTC GTT GCC Klf2fl/fl m control mice were immunized by injecting 20 l of 1 mg/ml OVA GGT CCA-39, reverse, 59-ACC AGC GCA GCG ATA TCG TC-39; Klf2 mixed 1:1 with CFA (Sigma-Aldrich) into the hock with a 27-gauge needle forward, 59-ACA GAC TGC TAT TTA TTG GAC CTT AG-39, reverse, 59- as previously described (30). Draining lymph node cells were harvested CAG AAC TGG TGG CAG AGT CAT TT-39; Ifng forward, 59-AAC GCT from inguinal and popliteal lymph nodes 10 d after immunization. Lymph ACA CAC TGC ATC TTG G-39, reverse, 59-GCC GTG GCA GTA ACA node cells were cultured in 96-well flat-bottom plates at 2.5 3 105 cells/ GCC-39; Il17a forward, 59-GCT CCA GAA GGC CCT CAG A-39, reverse, well in complete DMEM supplemented with L-glutamine, sodium pyru- 59-AGC TTT CCC TCC GCA TTG A-39; Il4 forward, 59-ACA GGA GAA vate, MEM–nonessential amino acids, and 2-ME and rechallenged with GGG ACG CCA T-39, reverse, 59-GAA GCC CTA CAG ACG AGC TCA- m 100 g/ml OVA or left untreated (control) for either 2 or 4 d. For cell 39; Tbx21 forward, 59-CAA CAA CCC CTT TGC CAA AG-39, reverse, proliferation experiments, lymph node cells were loaded with CFSE as 59-TCC CCC AAG CAG TTG ACA GT-39; Rorc forward, 59-GGA GCC previously described (31) prior to Ag rechallenge. AAG TTC TCA GTC ATG AGA ACA CA-39, reverse, 59-GCC CTT GCA Serum lipid analysis CCC CTC ACA GGT-39; Gata3 forward, 59-AGA ACC GGC CCC TTA TCA A-39, reverse, 59-AGT TCG CGC AGG ATG TCC-39; Foxp3 for- Mouse serum cholesterol and triglycerides were quantified using the c501 ward, 59-GGC CCT TCT CCA GGA CAG A-39, reverse, 59-GCT GAT module of the Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, IN). CAT GGC TGG GTT GT-39. Levels of specific gene expression were More detailed lipoprotein analyses were performed using HPLC normalized to endogenous levels of Actb gene expression and normalized (LipoSEARCH; Skylight Biotech, Tokyo, Japan). against control groups or expressed as percentage Actb. The Journal of Immunology 4653

Acetylated LDL uptake deficiency on atherosclerosis in Ldlr2/2 mice. For this purpose, we transplanted bone marrow from Itgaxcre-Klf2fl/fl and Klf2fl/fl BMDCs were generated by culture of bone marrow with GM-CSF (20 ng/ml) 2/2 for 7 d. Cells were washed and treated with 1 or 10 mg/ml human Dil– control mice into Ldlr mice and placed them on a high-fat acetylated LDL (Thermo Fisher Scientific) for 4 h. Cells were stained with and high-cholesterol diet for 10 wk. Transplant of Itgaxcre- Abs and Dil–acetylated LDL uptake was quantified by flow cytometry. Klf2fl/fl bone marrow into Ldlr2/2 mice successfully ablated Multiplexed cytokine assays KLF2 expression in both BMDCs and BMDMs (Fig. 2A, 2B).

+ Loss of DC-KLF2 had no effect on the percentage of circulating CD4 T cells were purified from splenocyte preparations by MACS cell CD11c+MHC class II+ DCs, but there was a trend toward in- separation using anti-CD4 (L3T4) microbeads (Miltenyi Biotec) as per the + + manufacturer’s instructions and stimulated in vitro overnight (18 h) with creased CD11c Flt3 precursor conventional DCs (Fig. 2C, 2D), plate-bound anti-CD3 mAb (145-2C11; BioLegend). Supernatant cytokine suggesting that KLF2 deficiency does not drastically disrupt levels were analyzed by proprietary multiplex analysis using the mouse overall DC development and hematopoiesis. Interestingly, Th17 array 25-plex discovery array (Eve Technologies, Calgary, AB, Itgaxcre-Klf2fl/fl mice showed an unexpected and profound re- Canada, catalog no. MTH17-25-106). Supernatants from BMDCs treated overnight with LPS (Sigma-Aldrich) were also analyzed using the mouse duction in the total numbers of circulating leukocytes (Sup- cytokine array/chemokine array 31-plex discovery assay (Eve Technolo- plemental Fig. 3A, 3B). This was characterized by an almost 2- gies, catalog no. MD31). fold reduction in the number of , monocytes, and . A reduction in blood leukocytes, neutrophils, lym- Statistical analyses phocytes, and monocytes was also seen in native LDL receptor– All statistical analyses were performed using GraphPad Prism software expressing Cd11c-Cre+ Klf2fl/fl mice compared with Cd11c-Cre– 6 (GraphPad Software). Values are expressed as means SEM unless Klf2fl/fl control animals, albeit not as profound as that seen in the Downloaded from otherwise noted. Pairwise comparisons were performed using Student t tests or Mann–Whitney U tests (nonparametric data). Multiple compar- bone marrow chimeras (Supplemental Fig. 3A, 3B). Hypercholes- isons of means for matched subjects were performed using two-way terolemia enhanced the reduction in total leukocyte counts, although ANOVA followed by Sidak multiple comparison tests. Differences be- there was high-fat diet–induced monocytosis and eosinophilia in tween groups were considered significant at probability values ,0.05. Itgaxcre-Klf2fl/fl mice as well as in controls. Cell numbers in both and heart lymph node were also reduced in hypercholester- fl/fl Results olemic Itgaxcre-Klf2 mice compared with controls (Fig. 3C, 3D), http://www.jimmunol.org/ CD11c-specific cre-lox deletion of KLF2 enhances DC indicating a global KLF2-dependent leukopenia. costimulatory molecule expression Loss of KLF2 in CD11c-expressing cells promotes a more To determine the phenotype of KLF2 deletion in DCs, we generated proatherogenic inflammatory monocyte phenotype under Itgaxcre-Klf2fl/fl mice that lack KLF2 in CD11c-expressing cells hypercholesterolemic conditions and compared them to Klf2fl/fl mice without a Cre transgene. KLF2 mRNA expression in CD11c+MHC class II+ BMDCs from Although reduced numbers were noted previously fl/fl Itgaxcre-Klf2fl/fl mice was reduced .90% compared with expres- in Lysmcre-Klf2 mice (22), hypercholesterolemia-induced sion in control mice. Furthermore, Itgaxcre-Klf2fl/fl BMDCs were monocytosis has been well established as an underlying factor unresponsive to KLF2-enhancing statin/rapamycin treatment in murine models of atherosclerosis (33, 34). We therefore by guest on September 28, 2021 (Fig. 1A). Naive CD4+ T cells, which express high levels of KLF2, asked whether KLF2 deficiency influenced the inflammatory/ but not CD11c, showed no difference in KLF2 expression between nonclassical monocyte balance. Whereas total monocyte lev- fl/fl Itgaxcre-Klf2fl/fl and Klf2fl/fl control mice (Fig. 1B). An expected els were reduced in Itgaxcre-Klf2 mice, Ly-6C staining hi loss of KLF2 expression, however, was seen in peritoneal mac- showed an increased percentage of proatherogenic Ly-6C , rophages (Fig. 1C), as many of these cells also express CD11c inflammatory monocytes (Fig. 2E). This was perhaps related to + (Supplemental Fig. 2). Unlike BMDCs, Itgaxcre-Klf2fl/fl perito- a reduced percentage of CD11c monocytes, which make up the mid/lo neal macrophages maintained KLF2 responsiveness upon statin/ vast majority of Ly-6C nonclassical monocytes (Fig. 2F, rapamycin treatment (Fig. 1D), consistent with the interpretation 2G). In contrast to monocytes, splenic macrophages accounted fl/fl that many of these cells are CD11c2 macrophages, which retain for a higher fraction of total spleen cells in Itgaxcre-Klf2 mice functional Klf2 . versus control mice (Fig. 2H), whereas total splenic macrophage Because we found that rapamycin enhances KLF2 expression in numbers did not significantly differ (Fig. 2I, 2J). Taken together, DCs, and others have shown that rapamycin induces a tolerogenic these data show that KLF2 deletion in our model is restricted to DC phenotype that expresses lower levels of costimulatory markers CD11c-expressing myeloid cells and includes DCs as well as a CD40 and CD80/CD86 (32), we conducted in vitro studies to de- significant percentage of monocytes and macrophages that normally termine whether loss of DC-KLF2 enhances costimulatory mole- express CD11c. Furthermore, we observed both leukopenia and an hi cule expression in mature DCs. Itgaxcre-Klf2fl/fl BMDCs showed inflammatory Ly-6C skewed monocyte fraction in the Itgaxcre- fl/fl increased surface expression of both CD40 and CD86 following Klf2 mice, which, based on previous studies, should have op- LPS activation (Fig. 1E), consistent with a more activated, proin- posing effects on atherosclerosis. flammatory phenotype. Analyses of supernatants from these BMDC Loss of DC-KLF2 exacerbates atherosclerosis development cultures showed no difference in DC secretion of IL-1, TNF-a, 2/2 IL-12, or IL-10 (data not shown). Ldlr mice develop hypercholesterolemia and atherosclerosis when fed a high-fat/high-cholesterol diet, even after they are ir- CD11c-KLF2 depletion induces leukopenia that is exacerbated radiated and reconstituted with Ldlr+/+ bone marrow, due to a by hypercholesterolemia deficiency in hepatic lipoprotein uptake, and these mice have been Previous research has shown that LysM-Cre–specific loss of KLF2 used extensively to study the impact of immune inflammatory in macrophages and neutrophils triggers spontaneous basal mac- processes in atherosclerosis (35). Ldlr2/2 recipients of Itgaxcre- rophage activation, enhances microbe-induced macrophage acti- Klf2fl/fl bone marrow showed no difference in weight, total serum vation, and aggravates atherosclerosis (17, 22). We hypothesized cholesterol, or serum triglycerides after 10 wk of a proatherogenic that DC-KLF2 may function to suppress proatherogenic T cell diet, compared with recipients of control bone marrow (Fig. 3A, responses, and we therefore examined the effect of CD11c-KLF2 3C). However, Oil Red O analysis of aortic sinus lesions showed 4654 DC-KLF2 AND THE PROATHEROGENIC IMMUNE RESPONSE

enhanced atherosclerosis in the Itgaxcre-Klf2fl/fl group in both male and female mice (Fig. 3D). The percentage macrophage content of lesions as determined by MAC-3 staining did not differ between groups (Fig. 3E), suggesting that KLF2 deficiency in CD11c+ monocyte/macrophages does not influence plaque mac- rophage numbers. There was a significant increase in aortic root adventitial neutrophils (clone NIMP-R14) (Fig. 3F). Anti- neutrophil–stained cells colocalized with myeloperoxidase staining, suggesting that the stained cells were neutrophils rather than monocytes/macrophages (data not shown). The proathero- genic effect observed in our model is seen despite the fact that the development of atherosclerosis is usually accompanied by a marked monocytosis (34, 36). Instead, our hypercholesterolemic Itgaxcre-Klf2fl/fl recipients showed a comparatively blunted monocytosis and deepened lymphopenia compared with hy- percholesterolemic Klf2fl/fl control mice. Thus, it appears that the proinflammatory phenotype of KLF2-deficient DCs has a dominant proatherosclerotic effect over the net reduction in

circulating monocytes and other leukocytes that contributes to Downloaded from lesion growth. Increased intimal DC accumulation corresponds with reduced intimal CD4+ T cells and increased apoptosis within lesions To better understand how KLF2-deficient DCs could enhance le-

sion inflammation and growth, we analyzed the numbers and lo- http://www.jimmunol.org/ calization of lesional DCs and T cells. CD11c immunofluorescence staining of aortic sinus lesions showed that intimal DCs accu- mulate in significantly greater numbers in Itgaxcre-Klf2fl/fl lesions (Fig. 4A). Intimal DCs from Itgaxcre-Klf2fl/fl lesions also showed a different pattern of localization compared with control lesions, with more DCs residing deep within the lesion, beyond the sub- endothelial space where intimal DCs tend to reside. Interestingly, acetylated LDL uptake was not different between Itgaxcre-Klf2fl/fl

versus control DCs (Supplemental Fig. 4), consistent with the by guest on September 28, 2021 interpretation that the impact of DC-KLF2 deficiency on enhanced lesion development is indirectly related to effects on other cells. Immunostaining of CD4 showed a significant reduction in intimal, but not adventitial, CD4+ T cells in Itgaxcre-Klf2fl/fl lesions (Fig. 4B). The reduced lesional T cell numbers are consistent with the global lymphopenia observed in these mice. This is in contrast with previous studies that have shown that increased T cell numbers are associated with more lesion development (37). Sur- prisingly, we noted the loss of intimal, but not adventitial, T cells, which corresponded roughly with increased intimal DC accumu- lation, suggesting that interactions between KLF2-deficient DCs and T cells may be a root cause of reduced T cell numbers. In support of this interpretation, TUNEL staining showed enhanced intimal apoptosis within Itgaxcre-Klf2fl/fl mouse lesions (Fig. 4C), suggesting that the loss of DC-KLF2 results in enhanced in- flammation and cell death within lesions. Thus, even though proatherogenic T cell accumulation may be reduced in lesions, the inflammatory consequences of their early activation, and perhaps other inflammatory results of KLF2-deficient DCs, re- sult in more lesion development.

purified splenic naive CD4+ T cells (B), peritoneal macrophages (C), and untreated (NT) or simvastatin/rapamycin–treated (S/R) peritoneal macro- FIGURE 1. Cre-lox deletion of KLF2 selectively depletes Klf2 ex- phages (D). (E) CD40 and CD86 expression on LPS-matured CD11c+MHC pression from CD11c-expressing cells and enhances costimulatory mole- class II+ BMDCs was measured by flow cytometry. Histograms show cule expression in DCs following activation. (A) BMDCs were generated representative staining from four independent experiments. Bar graphs from Klf2fl/fl (Cntrl, open bars) or Itgaxcre-Klf2fl/fl (KO, filled bars) bone quantify CD40 and CD86 mean fluorescence intensity (MFI) normalized marrow, matured with LPS alone or LPS plus simvastatin/rapamycin, and to control. (A) n = 10; (B–D) n =3;(E) n =6.*p , 0.05, **p , 0.01, Klf2 mRNA was measured by qRT-PCR. Klf2 mRNA was measured in ***p , 0.001. The Journal of Immunology 4655 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Loss of KLF2 in DCs promotes a more proatherogenic inflammatory monocyte phenotype under hypercholesterolemic conditions. Itgaxcre- Klf2fl/fl (KO) or Klf2fl/fl (Cntrl) bone marrow was transplanted into Ldlr2/2 recipients and fed a high-fat diet for 10 wk. (A and B) qRT-PCR for Klf2 mRNA was performed on BMDCs and BMDMs (BM-Mɸ) generated from Cntrl and KO bone marrow recipients following sacrifice. (C and D) Flow cytometry was performed on blood to determine circulating levels of mature DCs (C) and DC precursors (D). (E–G) Circulating monocytes were stained for Ly-6C and CD11c expression. (E and F) Percentages of Ly-6Chi and CD11c+ monocytes, respectively. (G) Representative flow cytometry plot for Ly-6C and CD11c staining. (H–J) Splenocytes were stained for macrophage markers CD11b and F4/80. (H) Macrophages as percentage of total splenocytes. (I) Splenic macrophage numbers calculated from total splenocyte counts. (J) Representative splenic F4/80, CD11b flow cytometry plot. (A) n = 11; (B) n =6;(C–J) n = 10–12. *p , 0.05, **p , 0.01, ****p , 0.0001.

DC-KLF2 modulates T cell activation in hypercholesterolemic development or T cell activation. CD25 and Foxp3 staining of mice splenic CD4+ T cells showed that Itgaxcre-Klf2fl/fl bone marrow To explain how atherosclerosis can increase in the context of recipients were as capable of maintaining splenic Tregs as controls + 2 lymphopenia and reduced monocyte numbers, we investigated (Fig. 6G), but they had more activated CD25 Foxp3 T cells splenocytes from the hypercholesterolemic Ldlr2/2 mice trans- (Fig. 5H, 5I). planted with either Itgaxcre-Klf2fl/fl or Klf2fl/fl control bone mar- To further explore whether DC-KLF2 influences T cell activation + row. Flow cytometry analyses of spleen show that, as seen in or Th subset lineage commitment, splenic CD4 T cells from the fl/fl fl/fl circulating leukocytes, hypercholesterolemic Itgaxcre-Klf2fl/fl Itgaxcre-Klf2 or Klf2 control bone marrow recipients were bone marrow recipients had markedly reduced numbers of T cells, restimulated with anti-CD3 Abs. qRT-PCR of Th lineage–specific but not B cells (Fig. 5A, 5C). This reduction in T cell numbers was mRNA from these experiments showed no difference in Th1- seen in both CD4+ and CD8+ T cell compartments (Fig. 5D, 5F), restricted Tbx21, Th17-restricted Rorc, or Treg-restricted Foxp3 suggesting that some aspect of T cell development, activation, or expression, whereas expression of Th2-restricted Gata3, which is maintenance is disrupted in the absence of DC-KLF2. We next usually associated with reduced atherosclerosis, was increased in sought to determine whether loss of DC-KLF2 affects Treg the Itgaxcre-Klf2fl/fl group (data not shown). Stimulated CD4+ 4656 DC-KLF2 AND THE PROATHEROGENIC IMMUNE RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Loss of CD11c-KLF2 aggravates atherosclerosis. Klf2fl/flCd11c-Cre2/2 (Cntrl) or Klf2fl/flCd11c-Cre+/2 (KO) bone marrow was transplanted into 12 male and 12 female Ldlr2/2 recipients and fed a high-fat diet for 10 wk. (A) Animal weight at sacrifice. (B and C) Blood serum was taken at sacrifice and lipid levels were measured to determine total cholesterol and triglyceride levels. (D–F) Frozen sections from aortic sinus were obtained and atherosclerotic lesions from the three-valve region were stained with Oil Red O for neutron lipid (D), by immunohistochemistry with Abs specific for macrophage marker MAC-3 (E), or by anti-neutrophil Ab (NIMP-R14) (F). Nuclei were counterstained with hematoxylin. Isotype controls for MAC-3 and NIMP-R14 staining were negative (not shown). Bar graphs indicate lesion area as percentage of lumen for male or female mice. Scale bars, 500 mm. (A–C) n = 12; (D) n = 20; (E), n = 12; (F), n =6.*p , 0.05. The Journal of Immunology 4657 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. KLF2-deficient DCs accumulate within intimal lesions corresponding with intimal CD4+ T cell loss. Frozen sections of aortic sinus were obtained from Ldlr2/2 recipients of Klf2fl/fl (Cntrl) or Itgaxcre-Klf2fl/fl (KO) bone marrow following 10 wk of high-fat diet and stained for CD11c (A)or CD4 (B). (A) Micrographs show CD11c staining (red), nuclear staining by DAPI (blue), and elastic laminae autofluorescence (green). Scale bars, 200 mm; inset scale bars, 50 mm. Bar graph shows CD11c stain index (no. of red pixels/no. of blue pixels) for Cntrl (open bars) or KO (filled bars) bone marrow recipients. (B) Micrographs show CD4 staining (red) or isotype control. Nuclei are counterstained with hematoxylin. Scale bars, 200 mm; inset scale bars, 50 mm. Bar graph shows number of intimal (Int) and adventitial (Adv) CD4 T cells per section and indicate lesion area as percentage of lumen for male or female mice. (C) Micrographs show TUNEL staining or negative control. Nuclei are counterstained with hematoxylin. Scale bars, 200 mm. (A–C) n = 10–12. *p , 0.05, ****p , 0.0001. 4658 DC-KLF2 AND THE PROATHEROGENIC IMMUNE RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Pan-CD11c KLF2 deficiency enhances T cell activation while reducing overall T cell numbers in hypercholesterolemic mice. Splenocytes from Klf2fl/fl (Cntrl) or Itgaxcre-Klf2fl/fl (KO) bone marrow recipients were stained for CD3, B220, CD4, and CD8 and analyzed by flow cytometry. (A and B) Total numbers of CD3+ T cells and B220+ B cells per spleen. (C) Representative FACS plots show CD3 and B220 staining of splenocytes. (D and E) Total numbers of CD4+ and CD8+ T cells per spleen. (F) Representative FACS plots of CD4 and CD8 staining gated on CD3+ splenocytes. (G–I) Splenocytes were also stained for CD4, CD25, and Foxp3. (G) Percentage of CD4+ T cells that are CD25hiFoxp3+ Tregs. (H) Percentage of activated CD4+ T cells (CD4+CD25+Foxp32). (I) Representative staining and gating of CD4+ T cell data shown in (G) and (H). (J) CD4+ splenocytes were isolated by MACS and stimulated overnight with anti-CD3 Ab. Graph shows supernatant cytokine levels determined by multiplex cytokine array. (K–M) RNA was also taken from anti-CD3–stimulated cultures and analyzed by qRT-PCR for expression of Ifng (K), Il17a (L), and Il4 (M). (A–H) n = 12; (J) n =6;(K–M) n = 10. *p , 0.05, **p , 0.01, ****p , 0.0001.

T cell culture supernatants showed a 2-fold increase in IL-2 pro- data support the cytokine data, showing increased expression duction in Itgaxcre-Klf2fl/fl bone marrow recipient mice, as well as of proinflammatory, proatherogenic Th1 cytokine Ifng in CD4+ significantly increased IL-4, IL-5, TNF-a, and soluble CD40L T cell cultures from Itgaxcre-Klf2fl/fl mice, whereas Il17a mRNA production (Fig. 5J). The increased IL-2 production is consistent expression was reduced (Fig. 5K, 5L). Il4 mRNA showed a with the enhanced activation-induced cell death (AICD) we ob- nonsignificant trend toward increased expression in Itgaxcre- served, because this cytokine is known to enhance Fas-FasL–induced Klf2fl/fl samples in line with the increased Gata3 expression and death (38). IFN-g secretion was also enhanced in Itgaxcre-Klf2fl/fl IL-4 and IL-5 secretion (Fig. 5M). Taken together, these data cells, although this trend was not significant, whereas Th17-produced show a highly activated CD4+ T cell phenotype in the absence of IL-17 and Treg-produced IL-10 showed no difference. qRT-PCR DC-KLF2, which surprisingly favors both Th1 and Th2 T cell The Journal of Immunology 4659 Downloaded from

fl/f fl/fl

FIGURE 6. KLF2-deficient DCs promote enhanced T cell proliferation and AICD (apoptosis). Klf2 (Cntrl) or Itgaxcre-Klf2 (KO) mice were im- http://www.jimmunol.org/ munized in the hock with whole OVA and CFA and draining lymph node cells were harvested 10 d later. (A and B) Draining lymph node cells from OVA- immunized mice were loaded with CFSE (CFDA-SE) and rechallenged with OVA or left unstimulated. After 4 d, cultures were harvested and stained with Abs against CD90.2 and CD4, as well as the viability dye 7-AAD. (A and B) Proliferation index for CD4+ and CD8+ T cells measured by CFSE dilution in response to OVA (black lines in histograms) over unstimulated baseline (dashed gray lines). (C and D) Draining lymph node cells from OVA-immunized mice were rechallenged with OVA or left unstimulated (No Ag) and stained with CD90.2, CD4, CD8, annexin V, and 7-AAD after 2 d. Shown are the sums of early (Q3) and late (Q2) apoptotic CD4+ (C) and CD8+ (D) T cells. Representative FACS plots are shown on the right for two independent experiments. (A and B) n =8;(C and D) n =6.*p , 0.05, **p , 0.01. CFDA-SE, CFSE.

activity, but not Th17 cells. However, the lack of a compensatory Discussion by guest on September 28, 2021 increase in Treg IL-10 production or Treg numbers implies that An important role for DCs in the modulation of inflammation has Treg responses may not keep pace with enhanced T cell activation been established for atherosclerosis (39). Evidence has been found in response to hypercholesterolemia, perhaps explaining how showing that DCs can both promote and protect against athero- plaque burden increased in Itgaxcre-Klf2fl/fl mice despite reduced sclerosis, depending either on lineage or cytokine milieu. Thus, a monocyte and T cell numbers. better understanding of the molecular mechanisms that influence DC control of pro- and anti-atherogenic T cell responses is needed Loss of DC-KLF2 enhances T cell proliferation and apoptosis to identify future molecular targets of drug or gene therapies. We next examined whether T cells from mice with KLF2-deficient However, the role of KLF2 in modulating how DCs influence DCs showed enhanced activation in vitro and whether KLF2- atherosclerosis has not been previously explored. deficient DCs induce higher rates of apoptosis in activated We first looked at how KLF2 influences DCs by performing T cells. Itgaxcre-Klf2fl/fl and control mice were immunized in the phenotypic and functional studies in Itgaxcre-Klf2fl/fl and control hock with OVA protein in CFA. Draining lymph nodes were BMDCs. Our data show that lack of DC-KLF2 enhanced DC harvested 10 d later and restimulated in vitro with OVA. OVA- expression of costimulatory molecules CD40 and CD86 (Fig. 1E). specific T cell proliferation was tested by CFSE dilution in 4 d This is consistent with a study that showed that silencing KLF2 cultures. Both CD4+ and CD8+ T cells from Itgaxcre-Klf2fl/fl mice expression in human monocyte-derived DCs enhanced CD80 (40). showed significantly enhanced Ag-induced proliferation compared A previous report describing Lyz2cre-specific deletion of KLF2 in with controls, consistent with enhanced in vivo naive T cell myeloid cells showed spontaneous myeloid cell activation in the priming by KLF2-deficient DCs (Fig. 6A, 6B). Apoptosis was absence of myeloid KLF2 (17). However, Itgaxcre-Klf2fl/fl deletion measured in 2 d cultures via annexin V staining, which showed did not influence cytokine production in BMDCs prior to and that both CD4+ (Fig. 6C) and CD8+ (Fig. 6D) T cells from following LPS-induced activation (data not shown). Thus, al- Itgaxcre-Klf2fl/fl mice had increased rates of apoptosis compared though KLF2 has been shown to function as a critical factor in with T cells from control mice following in vitro Ag rechallenge. maintaining cellular quiescence in other cell types (15, 17, 18, 41– However, there was no significant difference in cell death between 43), our cytokine data suggest that in DCs, KLF2 plays a more the two groups in the absence of OVA. These data show that al- subtle role, controlling instead the “volume” of context-dependent though DC-KLF2 is not required for Treg development or main- DC activation rather than suppressing spontaneous DC activation. tenance, it does play a role in modulating effector T cell This interpretation is further supported by the absence of a activation, expansion, and AICD. The enhanced T cell death is pronounced autoimmune phenotype in our Itgaxcre-Klf2fl/fl mice, consistent with fewer T cells in the blood, spleen, and lesions of as we did not observe spontaneous tissue-specific or systemic Itgaxcre-Klf2fl/fl mice despite the increased ability of KLF2-deficient inflammation or increased mortality. CD40 signaling between DCs DCs to activate T cells. and T cells has been shown to enhance several aspects important 4660 DC-KLF2 AND THE PROATHEROGENIC IMMUNE RESPONSE to T cell stimulation, including upregulation of ICAM-1 that Immunostaining of lesions showed increased numbers of intimal serves to stabilize the DC/T cell interaction, increased expression CD11c+ cells present in Itgaxcre-Klf2fl/fl plaques (Fig. 5). This is of CD80 and CD86 that provides costimulatory signals needed for perhaps explained by our finding of increased circulating precur- T cell activation (44), increased expression and stability of MHC sor conventional DCs, which serve as precursors for tissue- class II–peptide complexes (45, 46), prolonged DC survival (47, resident DCs (54) (Fig. 2D). It is possible that some of the 48), and prolonged Ag persistence following LPS-induced matu- CD11c staining is on macrophages, which we cannot distinguish ration (46, 49–51). Taken together, these findings indicate that from CD11c+ DCs in the lesions, but both cell types should be DC-KLF2 deficiency may promote a more proinflammatory DC competent at activating T cells within the lesion, and both would phenotype by enhancing CD40-CD40L signaling between DCs be susceptible to CD11c-Cre–driven Klf2fl/fl deletion. Detailed and T cells, independent of DC cytokine production. CD40- electron scanning microscopy studies of human atherosclerotic CD40L signaling, in turn, likely enhances DC survival, Th cell lesions have shown that immature vascular DCs lay in contact (CD4+) differentiation, and priming cytotoxic CD8+ T cell re- with the basal side of the endothelial layer before activating and sponses. This establishes our Itgaxcre-Klf2fl/fl model as pheno- joining a webbed network of subendothelial leukocytes. These typically distinct from previous myeloid lineage tissue-specific activated DCs then continue to migrate deeper into the growing KLF2-KO models. lesion and show morphological evidence of greater inflammatory KLF2 expression was also reduced in CD11c-expressing mono- activity as disease progresses (55–57). As shown in Fig. 4A, in- cytes and macrophages (Fig. 1C, 1D). A monocyte/macrophage creased numbers of CD11c+ staining cells appear deeper within phenotype was also seen in our atherosclerosis model where intimal lesions, which points to a more activated, inflammatory 2 2 Ldlr / recipients of Itgaxcre-Klf2fl/fl bone marrow showed reduced DC phenotype, consistent with our in vitro data showing enhanced Downloaded from KLF2 expression in BMDCs and BMDMs as well (Fig. 2A, 2B). maturation of KLF2-deficient DCs and increased expression of Hypercholesterolemic recipients of Itgaxcre-Klf2fl/fl bone marrow costimulatory molecules. Reduced intimal CD4+ T cell numbers showed a significantly increased skewing of the monocyte com- coupled with increased lesional TUNEL staining in recipients of partment toward an inflammatory phenotype compared with con- Itgaxcre-Klf2fl/fl bone marrow also support the theory of enhanced trols, which can be explained by the fact that most Ly-6Clo/mid DC-mediated T cell activation in vivo, as lesional T cell activation

nonclassical monocytes express CD11c (Fig. 2E, 2G). Prior studies is a well-known contributor to cell death and necrotic core for- http://www.jimmunol.org/ have shown that inflammatory macrophages express less KLF2 mation (2, 3). than do nonclassical macrophages (52, 53). From our data, it is It is paradoxical, then, that atherosclerosis was enhanced in possible that many CD11c-expressing Ly-6Clo/mid nonclassical Itgaxcre-Klf2fl/fl bone marrow recipients in the context of reduced monocytes lose KLF2 and upregulate Ly-6C, becoming inflam- circulating and splenic T cells (Fig. 5). Although prior studies of matory Ly-6Chi monocytes, as Itgaxcre-Klf2fl/fl bone marrow LysM-Cre Klf2fl/fl mice reported proinflammatory changes in recipients had increased numbers of CD11c+Ly-6Chi, but not mainly monocyte/macrophage and neutrophil populations (17, CD11c2Ly-6Chi, compared with controls (Fig. 2G). This 22), our studies of hypercholesterolemic Itgaxcre-Klf2fl/fl mice proinflammatory skewing of the monocyte compartment in Itgaxcre- showed profound changes in T cell responses. Most notably, there Klf2fl/fl bone marrow–recipient mice was accompanied by a para- were more activated CD25+ splenic CD4+ T cells in Itgaxcre- by guest on September 28, 2021 doxical reduction in circulating monocyte numbers (Fig. 4A, 4B), Klf2fl/fl bone marrow recipients, whereas Treg responses remained which corresponds with findings in previous studies of myeloid- unchanged compared with control (Fig. 5G, 5I). Purified splenic specific KLF2-KO mice (17, 22). This monocyte loss was accom- CD4+ T cells from Itgaxcre-Klf2fl/fl mice produced increased panied by an even greater reduction in lymphocytes in both levels of both Th1 and Th2 cytokines upon restimulation with hypercholesterolemic Itgaxcre-Klf2fl/fl bone marrow recipients and anti-CD3 mAb, including IL-2, IFN-g, TNF-a, IL-4, and IL-5 normocholesterolemic Itgaxcre-Klf2fl/fl mice maintained on chow (Fig. 5J, 5M). Meanwhile, Th17 and Treg responses were either diet (Supplemental Fig. 3A, 3B). Reduced leukocyte numbers reduced or unchanged. One possible explanation for increased Th1 were also found in spleen and heart-draining lymph nodes of and Th2 responses in the absence of a correspondingly increased hypercholesterolemic mice (Supplemental Fig. 3C, 3D), suggest- Treg response comes from prior work in a Leishmania infection ing that this cell loss occurs in multiple compartments. These model, which showed that CD40lo DCs are more effective at in- findings are distinct from those previously reported in myeloid- ducing Tregs, whereas CD40hi DCs primarily induce effector restricted KLF2-KO models and highlight the Itgaxcre-Klf2fl/fl T cell responses (58). These findings indicate that KLF2-deficient phenotype as a unique indicator of KLF2 function in DCs. DCs enhance a broad spectrum of T cell activation. The major finding in this study is that, despite significantly We confirmed that there was enhanced CD4+ and CD8+ T cell reduced monocyte and lymphocyte numbers, loss of CD11C-KLF2 activation by KLF2-deficient DCs in independent experiments enhanced atherosclerotic plaque burden in hypercholesterolemic by immunizing Itgaxcre-Klf2fl/fl or control mice with OVA and mice without enhancing body weight or blood cholesterol levels measuring OVA-induced proliferation of primed cells ex vivo beyond those of control mice (Fig. 4). Myeloid KLF2 depletion in (Fig. 6A, 6B). T cell apoptosis following Ag rechallenge was also LysM-Cre mice was previously shown to enhance atherosclerosis increased in Itgaxcre-Klf2fl/fl mice (Fig. 6C, 6D). Together with in Ldlr2/2 mice (22). However, that study found a mechanism of increased T cell activation and IL-2 production, enhanced T cell action involving enhanced neutrophil and macrophage adhesion apoptosis indicates that the reduced global T cell numbers seen in and homing to atherosclerotic plaques via enhanced expression of hypercholesterolemic Itgaxcre-Klf2fl/fl bone marrow recipients CD11b. In our model, Itgaxcre-Klf2fl/fl plaques had equal amounts likely occurs at least in part due to AICD. AICD has long been of macrophages as shown by MAC-3 staining as control plaques known to be driven by IL-2 binding to its high-affinity receptor, (Fig. 3E), despite CD11c+ macrophage populations in spleen and CD25, which was also increased on the surface Itgaxcre-Klf2fl/fl peritoneum expressing significantly more CD11b as well (data not T cells (59–61). shown). Thus, it is likely that reduced monocyte numbers may in Collectively, our findings highlight the previously unknown part be due to enhanced lymphoid and peripheral tissue recruit- importance of KLF2 in regulating T cell inflammatory responses. ment of monocytes and a higher rate of tissue macrophage acti- DCs lacking KLF2 express higher levels of costimulatory mole- vation and death (Figs. 2H, 4). cules such as CD40 and CD86 upon activation and “overactivate” The Journal of Immunology 4661

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