Two Categories of Biologic Effects Induced by IL-7 on Human T Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3303-3303
Author(s):  
Thomas P. Krupica ◽  
Chiguang Feng ◽  
Crystal L. Mackall ◽  
Terry J. Fry
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
Dose Response ◽  
Low Dose ◽  
High Dose ◽  
T Cell Homeostasis ◽  
Dose Effects ◽  
Biologic Effects ◽  
Low Dose Effects

Abstract Background: IL-7 is constantly available, most mature T cells express the IL-7R complex, and IL-7 signaling is required for mature T cell survival, thus implicating IL-7 as a trophic cytokine. However, since IL-7Rα (CD127) expression on T cells is dynamically regulated in response to activation and IL-7 exposure, increased IL-7 levels present during lymphopenia augment homeostatic expansion, and IL-7 therapy induces dramatic alterations in T cell homeostasis, it can be inferred that IL-7’s effects on T cells critically depends on concentration. We postulated therefore, that dose response effects may be central to IL-7’s capacity to modulate T cell homeostasis. Methods: We evaluated dose response effects of IL-7 on naïve vs. memory CD4+ and CD8+ mature human T cells in vitro using five distinct biologic effects of IL-7 as endpoints: Stat5a phosphorylation, co-stimulation of anti-CD3 mediated proliferation, Bcl-2 up-regulation, CXCR4 up-regulation, and IL-7Rα down-regulation. Using CD45RO based immunomagnetic bead separation (Miltenyi), fresh human peripheral blood T cells were separated into naïve (CD45RO−) vs. memory (CD45RO+) subsets, then cultured for 5 days with increasing concentrations of IL-7 (0.1ng/ml – 10ng/ml). On day 5, cells were analyzed by flow cytometry for the endpoints noted. Intracellular pathways implicated in IL-7 signaling on T cells were probed using PI3K (LY294002) and mTOR (Rapamycin) inhibitors. Results: The biologic effects of IL-7 on mature T cells can be grouped into two categories. The first category consists of Stat5a phosphorylation and co-stimulation for proliferation. These effects occur at very low doses (0.1ng/ml) with gradually increasing percentages of cells responding with increasing doses. These responses appear to reflect receptor occupancy by the IL-7 molecule since subsets with higher IL-7Rα receptor expression show proliferative effects at lower IL-7 doses. Further, the proliferative effects of IL-7 are fully inhibited by either LY294002 (10μM) or Rapamycin (10ng/ml). In contrast to IL-7’s low dose effects, Bcl-2 and CXCR4 up-regulation, and IL-7Rα down-regulation can be grouped into a second category of effects that occur only in response to high dose IL-7 (10ng/ml). High dose effects occur in an “all or nothing” pattern with T cell subsets bearing low levels of IL-7Rα expression demonstrating the same dose response as subsets with high IL7Rα expression. Furthermore, high dose effects of IL-7 utilize differential signaling pathways compared to the low dose effects, as they are not inhibited by either LY294002 or Rapamycin. Conclusions: We have identified two categories of IL-7 effects on mature T cells. Low dose effects, which are primarily involved in co-stimulation for proliferation and PI3K/mTOR dependent, and are likely to be highly modulated by receptor regulation and small changes in IL-7 availability. Then in contrast, high dose effects including Bcl-2, CXCR4 and IL-7Rα modulation, which utilize separate signaling pathways as they are not PI3K/mTOR dependent. Whether high dose effects of IL-7 reflect signaling through a separate, low affinity IL-7R is currently under investigation. These results demonstrate previously unrecognized distinctions in IL-7 signaling pathways, and may help to explain why substantial alterations in T cell homeostasis occur when IL-7 is elevated during lymphopenia despite IL-7’s constant availability in a lymphoreplete environment.

scholarly journals CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by Recombinant Listeria monocytogenes during Secondary Infection

2002 ◽  
Vol 70 (1) ◽  
pp. 153-162 ◽  
Author(s):  
Amy R. Tvinnereim ◽  
Sara E. Hamilton ◽  
John T. Harty
Keyword(s):  
T Cells ◽  
Listeria Monocytogenes ◽  
T Cell ◽  
Cell Response ◽  
Low Dose ◽  
Recombinant Antigen ◽  
T Cell Response ◽  
Vector System ◽  
High Dose ◽  
The Impact

ABSTRACT Understanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuated actA-deficient Listeria monocytogenes, on the CD8+-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinant L. monocytogenes. Challenges of immune mice with actA-deficient and with wild-type recombinant L. monocytogenes generated similar numbers of CD8+ T cells specific for the NP118-126 epitope. High-dose immunization with actA-deficient L. monocytogenes resulted in substantial numbers of CD8+ T cells specific for the L. monocytogenes LLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinant L. monocytogenes resulted in rapid control of the infection and decreased CD8+-T-cell responses against both the secreted and nonsecreted form of the recombinant antigen compared to the response of naïve mice. In contrast, mice immunized with a low dose of actA-deficient L. monocytogenes had ∼10-fold fewer effector and memory T cells specific for LLO91-99 and a substantially higher CD8+-T-cell response against the recombinant antigen after challenge with recombinant L. monocytogenes. Although mice immunized with low-dose actA-deficient L. monocytogenes had a substantial recall response to LLO91-99, which reached the same levels by 5 to 7 days postchallenge as that in high-dose-immunized mice, they exhibited decreased ability to control L. monocytogenes replication. Thus, the level of antivector immunity impacts the control of infection and efficiency of priming responses against new antigens introduced with the same vector.

The Absence of Vasoactive Intestinal Peptide Augments Allo-Reactivity and the Anti-Viral Response in Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3267-3267
Author(s):  
Lauren T. Southerland ◽  
Jian-Ming Li ◽  
Sohrab Hossain ◽  
Cynthia Giver ◽  
Wayne Harris ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Low Dose ◽  
T Cell Response ◽  
High Dose ◽  
Mcmv Infection

Abstract Background: The severe morbidity and mortality associated with bone marrow transplantation (BMT) is caused by uninhibited immune responses to alloantigen and suppressed immune responses to pathogens. Vasoactive Intestinal Peptide (VIP) is an immunomodulatory neuropeptide produced by T-cells and nerve fibers in peripheral lymphoid organs that suppresses immune responses by induction of tolerogenic dendritic cells. In order to determine the immunoregulatory effects of VIP, we examined T-cell immune responses to allo- and viral-antigens in VIP knockout (KO) mice and mouse BMT recipients of hematopoietic cells from VIP KO donors. Methods: VIP KO mice and VIP WT littermates were infected with lethal or sub-lethal doses (5 × 104− 5 × 105 PFU) of murine cytomegalovirus (mCMV) and the T-cell response to viral antigen was measured by flow cytometry for mCMV peptide-MHC class 1-tetramer+ CD8+ T-cells. We transplanted 5 × 106 BM plus 1 × 106 splenocytes (SP) either from VIP KO or VIP WT donors in an C57BL/6 to F1(BL/6 × Balb/c) allo-BMT model and assessed survival, GvHD, donor T-cell expansion, chimerism, and response to mCMV vaccination and mCMV infection. Results: B-cell, αβ and γδ T-cell, CD8+ T-cell, CD11b+ myeloid cell, and dendritic cell numbers were equivalent between VIP KO and WT mice, while VIP KO mice had higher number of CD4+ and CD4+CD62L+CD25+ T-cells. Non-transplanted VIP KO mice survived mCMV infection better compared to VIP WT, with a brisker anti-viral T-cell response in the blood. In the allogeneic BMT setting, recipients of VIP KO BM plus VIP KO SP had more weight loss and lower (40%) 100 day post-transplant survival compared to the recipients of VIP KO BM plus WT SP (80% survival), recipients of WT BM plus KO SP (100% survival), and recipients of WT BM plus WT SP (80% survival). Recipients of VIP KO grafts had a significantly greater anti-mCMV response that peaked four days earlier than the tetramer response of mice transplanted with WT cells. This increased anti-viral response to vaccination correlated with a greater and more rapid T-cell response to secondary viral challenge. Conclusions: These experiments suggest that the absence of all VIP in the body, or the absence of VIP in a transplanted immune system, enhances anti-viral immunity and allo-immune responses. Modulation of the VIP pathway is a novel method to regulate post-transplant immunity. Figure 1: VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day. Figure 1:. VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day.

The marginalization of hormesis

2000 ◽  
Vol 19 (1) ◽  
pp. 32-40 ◽  
Author(s):  
E J Calabrese ◽  
L A Baldwin
Keyword(s):  
Dose Response ◽  
Low Dose ◽  
Close Association ◽  
Modern Medicine ◽  
High Dose ◽  
Multiple Factors ◽  
Toxicological Data ◽  
Dose Effects ◽  
Guilt By Association ◽  
Carry Over

Despite the substantial development and publication of highly reproducible toxicological data, the concept of hormetic dose-response relationships was never integrated into the mainstream of toxicological thought. Review of the historical foundations of the interpretation of the bioassay and assessment of competitive theories of dose-response relationships lead to the conclusion that multiple factors contributed to the marginalization of hormesis during the middle and subsequent decades ofthe 20th century. These factors include: (a) the close-association of hormesis with homeopathy lead to the hostility of modern medicine toward homeopathy thereby creating a guilt by association framework, and the carry-over influence of that hostility in the judgements of medically-based pharmacologists/ toxicologists toward hormesis; (b) the emphasis of high dose effects linked with a lack of appreciation of the significance of the implications of low dose stimulatory effects; (c) the lack of an evolutionary-based mechanism(s) to account for hormetic effects; and (d) the lack of appropriate scientific advocates to counter aggressive and intellectually powerful critics of the hormetic perspective.

scholarly journals Interleukin-15 Increases Effector Memory CD8+ T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques

2005 ◽  
Vol 79 (8) ◽  
pp. 4877-4885 ◽  
Author(s):  
Yvonne M. Mueller ◽  
Constantinos Petrovas ◽  
Paul M. Bojczuk ◽  
Ioannis D. Dimitriou ◽  
Brigitte Beer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Low Dose ◽  
Effector Memory ◽  
High Dose ◽  
Immunodeficiency Virus ◽  
Interleukin 15

ABSTRACT Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 μg/kg of body weight, n = 3; high dose of IL-15, 100 μg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3− NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA−CD62L− and CD45RA+CD62L− effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.

scholarly journals Safety and Response of Incorporating CD19 Chimeric Antigen Receptor T Cell Therapy in Typical Salvage Regimens for Children and Young Adults with Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 684-684 ◽  
Author(s):  
Daniel W. Lee ◽  
Maryalice Stetler-Stevenson ◽  
Constance M. Yuan ◽  
Terry J. Fry ◽  
Nirali N Shah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Burden ◽  
Low Dose ◽  
High Dose ◽  
T Cell Therapy ◽  
High Burden ◽  
Car T Cells ◽  
Car T ◽  
Preparative Regimen

CD19 chimeric antigen receptor (CAR) T cells have shown significant promise in multiple early phase trials including our own (Lancet 385:517-28). We manufacture CAR T cells containing CD28 and CD3z domains in 7 days using a retroviral platform. Several challenges remain to its widespread use: 1) reduction in the incidence of grade 4 cytokine release syndrome (CRS) and 2) incorporation with standard salvage regimens. Here, we update our experience with 39 patients. In the first 21 patients we defined the maximally tolerated dose as 1x106 CAR T cells/kg, grade 4 CRS occurred in 16%, and noted that severity of CRS correlated with disease burden. We stratified the current cohort (n=18) by disease burden. Subjects 1-21 and subsequent patients with low burden disease (Arm 1: isolated CNS disease or <25% marrow blasts) received a low dose preparative regimen of fludarabine (25 mg/m2/day Days-4 to -2) and cyclophosphamide (900 mg/m2 Day-2). Those with high burden disease (Arm 2: ³25% marrow blasts, circulating blasts or lymphomatous disease) received a high dose regimen to reduce tumor burden prior to cell infusion in an attempt to decrease severity of CRS. Arm 2 regimens were individualized based on prior therapies and risk from comorbidities. FLAG (n=6), ifosfamide/etoposide per AALL0031 (IE; n=2) and high dose fludarabine (30 mg/m2/day Days -6 to -3) with cyclophosphamide (1200 mg/m2/day Days -4 and -3) (HD flu/cy; n=3) were used. All products in the second cohort met cell dose though contaminating monocytes tended to inhibit maximal growth and transduction (see companion abstract by Stroncek). All patients received 1x106 CAR T cells/kg. Using grading criteria and an algorithm for early intervention to prevent grade 4 CRS (Blood 124:188-95) no grade 3 and only 1 grade 4 (5.6%) CRS occurred. Having significant comorbidities, Pt 34 was electively intubated for airway protection, did not require vasopressors, and rapidly recovered after tocilizumab and steroids. A brief seizure occurred, though he had a history of seizures. None others in the current cohort had neurotoxicity. Using intent to treat analysis, the complete response (CR) rate was 59% overall and 61% in ALL. 13/16 (81%) low burden and 10/22 (46%) high burden ALL patients had a CR across both cohorts. Low burden patients treated on either cohort had similar CR rate of 8/10 (80%) and 5/6 (83%). Although not statistically significant and underpowered, 7/11 (64%) high burden patients treated with low dose flu/cy had a CR while 3/11 (27%) had a CR with high dose regimens. Specifically, 3/6 (50%) receiving FLAG achieved MRD-CR while none receiving IE or HD flu/cy responded. 8/8 with primary refractory ALL had MRD-CR regardless of disease burden or preparative regimen raising the prospect that T cell fitness in these patients was superior to others. Of the 20 patients achieving an MRD-CR, the median leukemia free survival (LFS) is 17.7 months with 45.5% probability of LFS beginning at 18 months. Only 3 did not have a subsequent hematopoietic stem cell transplant as their referring oncologist determined the risk of such was unacceptable. Two relapsed with CD19-leukemia at 3 and 5 months, while 1 remains in CR with detectable CAR T cells at 5 months. Reliance on multiple infusions of cells is problematic as 0/5 CD19+ patients receiving a second dose responded. Preclinical models have demonstrated that T cell exhaustion has a role in limiting the efficacy of CAR T cells. We evaluated CAR products and the T cells used to generate them for phenotypic markers of exhaustion and will present data evaluating the relationship between these and response. Our results demonstrate that CD19 CAR T cell therapy is safe and effective with aggressive supportive care and use of an early intervention algorithm to prevent severe CRS and provides a potential for cure in primary refractory ALL. Table. Patient Characteristics, Response, and Toxicity Pt Age/ Sex/Risk # Relapses Arm/Prep Regimen(if Arm 2) Marrow Blasts Response CRS Grade Pre-Therapy Post CAR 22 17M 3 1 20 0 MRD- 2 23 13M 2 2 IE 99 98 SD 0 24 12M MLL 2 1 8.5 3 CR 1 25 25F 1 2 FLAG 95 0 MRD- 2 26 4M DS 2 2 IE (60%) 89 NA PD 0 27 8F 2 2 FLAG 77 69 SD 0 28 4M 2 2 FLAG (60%) 99 99 PD 0 29 12M PR 1 0.15 0 MRD- 1 30 15M Ph+ CNS2 3 1 0.08 0 MRD- 1 31 22M 3 2 FLAG 97 99 SD 0 32 15M CNS2 3 2 FLAG 0.04 + Lymphoma 0 MRD- 2 33 6M PR 1 0.15 0 MRD- 0 34 14M DS 3 2 Arm 1 Flu/Cy 90 0 MRD- 4 35 25M 2 2 HD Flu/Cy 30 87 PD 2 36 6M 2 1 1.5 91 PD 0 37 4F MLL 1 2 HD Flu/Cy 90 99 SD 0 38 7M 1 2 HD Flu/Cy 99 99 SD 1 Disclosures Off Label Use: Off-label use of tocilizumab will be discussed in managing cytokine release syndrome.. Rosenberg:Kite Pharma: Other: CRADA between Surgery Branch-NCI and Kite Pharma. Mackall:Juno: Patents & Royalties: CD22-CAR.

scholarly journals Hormones and Endocrine-Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses

2012 ◽  
Vol 33 (3) ◽  
pp. 378-455 ◽  
Author(s):  
Laura N. Vandenberg ◽  
Theo Colborn ◽  
Tyrone B. Hayes ◽  
Jerrold J. Heindel ◽  
David R. Jacobs ◽  
...  
Keyword(s):  
Dose Response ◽  
Low Dose ◽  
Endocrine Disrupting Chemicals ◽  
Low Doses ◽  
Response Curves ◽  
Toxicological Studies ◽  
Endocrine Disrupting ◽  
Dose Effects ◽  
Dose Response Curves ◽  
Low Dose Effects

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of “the dose makes the poison,” because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

scholarly journals 578 CD8-targeted IL-2 drives potent anti-tumor efficacy and promotes action of tumor specific vaccines

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A607-A607
Author(s):  
Hussein Sultan ◽  
Kelly Moynihan ◽  
Yuang Song ◽  
Samuel Ameh ◽  
Ton Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Body Weight Loss ◽  
Tumor Rejection ◽  
High Dose ◽  
Therapeutic Effects ◽  
Toxicity Profile ◽  
Tumor Bearing

BackgroundIL-2 and currently available engineered variants are of interest for solid tumor treatment, but their efficacy and toxicity profiles remain suboptimal. These results reflect the pleiotropic signaling via IL-2 receptors on different cell types that may simultaneously drive desired and undesired responses. We hypothesized that restricting IL-2’s activity to CD8+ T cells would improve efficacy while also lowering its toxicity profile.MethodsWe developed a cis-targeted IL-2 that selectively acts on CD8+ T cells (CD8-IL2) and assessed its activity using the T3 progressor MCA sarcoma model, which was selected because (a) it is sensitive to anti-PD-1 therapy when tumors are small but develops insensitivity as tumor size increase, (b) rejection requires both CD4+ and CD8+ T cells and (c) rejection is dependent on tumor expression of two neoantigens: mItgb1 (MHC-II) and mLama4 (MHC-I).ResultsWhereas mice bearing 8-day T3 tumors had become insensitive to anti-PD-1 mediated tumor rejection, 90% of mice treated with single dose CD8-IL2 monotherapy rejected their tumors, while high dose IL-2 produced minimal efficacy. Efficacy occurred without body weight loss. These results suggest that CD8-IL2 can induce therapeutic effects at a time when tumors became insensitive to anti-PD-1. To assess this possibility in a more controlled manner, we used a tumor neoantigen vaccine model that depends on CD4+ T cell help for development of functional CD8+ T cells at both the priming stage in the lymph node as well as the effector stage at the tumor site. Mice bearing T3 tumors were vaccinated with a synthetic long peptide (SLP) containing the mLama4 neoepitope and either a high or low dose of an SLP containing the mItgb1 neoepitope. Whereas 85% of tumor bearing mice that received the vaccine containing mLama4 plus low dose mItgb1 SLP rejected their tumors, surprisingly none of the mice receiving high dose mItgb1 underwent tumor rejection. This high dose inhibition was reversed when CD8-IL2 was administered after high dose vaccination and at concentrations that had only modest activity in tumor bearing, non-vaccinated mice. With CD8-IL2 treatment, antigen specific T cells were expanded and displayed increased expression of activation-associated markers and reduced expression of exhaustion-associated markers.ConclusionsCD8-IL2 outperformed other forms of engineered IL-2 in anti-tumor efficacy, showed a significantly improved toxicity profile, and rescued deficient CD8 T cell responses resulting from poor CD4 help. In sum, we demonstrate high level antitumor efficacy and tolerability with a new form of targeted IL-2.Ethics ApprovalMice used in this study were between 8 and 12 weeks of age and were maintained in accordance with procedures approved by the Association for Assessment and Accreditation of Laboratory Animal Care and Accredited Animal Studies Committee of Washington University in St. Louis

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