Engraftment Fitness of Human Central Memory-Derived Effector CD8+ T Cells In Immunodeficient Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1019-1019
Author(s):  
Xiuli Wang ◽  
Berger Carolina ◽  
Stanley R. Riddell ◽  
ChingLam W Wong ◽  
Stephen Forman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Tumor Response ◽  
Memory T Cells ◽  
Effector T Cells ◽  
Caspase Activity ◽  
Effector Cells ◽  
Nog Mice

Abstract Abstract 1019 Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of T cell transfer as a therapy for patients. But the potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. The repertoire of memory T cells is heterogeneous with respect to phenotypic, functional, and epigenetic attributes. Memory T cells are divided into sub-populations of 1) effector memory (TEM) cells that distribute to tissue beds and exhibit immediate cytolytic effector functioning, and 2) central memory (TCM) cells that home to lymph nodes based on CD62L/CCR7 expression and are capable of extensive proliferative activity upon re-encountering antigen. Thus the cell-intrinsic programming of distinct memory T cell subtypes, such as TEM and TCM, likely dictate divergent fates of their derived effector cells. To address this important issue, a clear functional dichotomy between TCM- and TEM-derived CD8+ CTLs was recently delineated in a nonhuman primate model, where it was found that virus-specific CD8+ CTL clones derived from TCM, but not TEM precursors, establish persistent and functional memory following adoptive transfer. Here, we extended these studies to human effector T cells using CMV as antigen model system to investigate the engraftment of human CMVpp65-specific CD8+ effector T cells derived in vitro from either sort purified CD45RO+CD62L+ TCM or CD45RO+CD62L- TEM precursors in NOD/Scid IL-2RγCnull (NOG) mice. TCM-derived effector cells (TE(CM)) and TEM-derived effector cells (TE(EM)) were adoptively transferred (i.v) into NOG mice reconstituted with human IL-15 and T cell levels in circulation were evaluated at different time points by FACS. 20% CD8+ TE(CM) and 3% CD8+ TE(EM) were detected on day 14. Then after, engraftment of the CD8+ TE(CM) remained at a steady state of approx 2% of circulating mononuclear cells for 100 days while TE(EM) remained at or below the level of detection, indicating that TE(CM) were superior in their ability to engraft in response to IL-15 as compared to TE(EM) after adoptive transfer (P<0.05). The long-term (100 days) persisting CD8+ TE(CM), harvested from primary recipient mice were found to be capable of engrafting secondary recipients. TcR Vβ analysis of persisting cells demonstrated that CD8+ TE(CM) engraftment was polyclonal, suggesting that homeostatic engraftment fitness is a general feature of these cells. To delineate the mechanism(s) by which TE(CM) exhibit superior in vivo engraftment, TE(CM) and TE(EM) were first labeled with CFSE before in vivo administration. CFSE profiles appear that the TE(EM) proliferated more extensively than TE(CM) early after adoptive transfer as indicated by the percent of cells which diluted CFSE on day 9 (i.e., 80% vs. only 25%, respectively). However, using D2R cleavage as a measure of caspase activity as a surrogate for apoptosis, 5.8% of engrafting TE(CM) were positive for activated caspase activity compared to 31.6% of TE(EM), suggesting that in NOG mice both CD8+ TE(CM) and TE(EM) proliferate in response to IL-15 whereas TE(CM) are intrinsically resistant to caspase activation and apoptosis. We also evaluated the antigen specific responsiveness of engrafted cells. Weekly infusions of irradiated pp65+/A2+ LCL as antigen significantly augmented the levels of circulating CD8+ TE(CM) as compared to no antigen stimulation (P<0.05), whereas CD8+ TE(EM) did not respond to antigen challenge. Moreover, when CMVpp65 specific CD8+ TE(CM) or TE(EM) were infused into CMVpp65+ tumor bearing mice, tumor cells progressed in mice receiving TE(EM) at a rate similar to untreated control mice over a ten day observation period, whereas TE(CM) significantly controlled tumor progression (P<0.05), indicating that CD8+ TE(CM) but not TE(EM) are able to mediate an anti-tumor response. Together these studies confirm that human CD8+ effector T cells derived from TCM precursors are capable of persistence after infusion, can proliferate in in vivo in response to antigen, can mediate an anti-viral or anti tumor response, and are likely the preferred T cells for antigen specific anti-tumor adoptive T cell therapy . Disclosures: No relevant conflicts of interest to declare.

scholarly journals H-2 restriction of virus-specific T-cell-mediated effector functions in vivo. II. Adoptive transfer of delayed-type hypersensitivity to murine lymphocytic choriomeningits virus is restriced by the K and D region of H-2.

1976 ◽  
Vol 144 (3) ◽  
pp. 776-787 ◽  
Author(s):  
R M Zinkernagel
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Gamma Globulin ◽  
Effector T Cells ◽  
Delayed Type Hypersensitivity ◽  
T Effector Cells ◽  
Immune Lymphocytes ◽  
D Region

In mice, primary footpad swelling after local infection with lymphocytic choriomeningitis virus (LCMV) and delayed-type hypersensitivity (DTH) adoptively transferred by LCMV immune lymphocytes are T-cell dependent. Nude mice do not develop primary footpad swelling, and T-cell depletion abrogates the capacity to transfer LCMV-specific DTH. Effector T cells involved in eliciting dose-dependent DTH are virus specific in that vaccinia virus-immune lymphocytes could not elicit DTH in LCMV-infected mice. The adoptive transfer of DTH is restricted to H-2K or H-2D compatible donor-recipient combinations. Distinct from the fowl-gamma-globulin DTH model, I-region compatibility is neither necessary nor alone sufficient. Whatever the mechanisms involved in this K- or D-region associated restriction in vivo, it most likely operates at the level of T-cell recognition of "altered self" coded in K or D. T cells associated with the I region (helper T cells and DTH-T cells to fowl-gamma-globulin) are specific for soluble, defined, and inert antigens. T cells associated with the K and D region (T cells cytotoxic in vitro and in vivo for acute LCMV-infected cells, DTH effector T cells, and anti-viral T cells) are specific for infectious, multiplying virus. The fact that T-cell specificity is differentially linked with the I region or with the K and D regions of H-2 may reflect the fundamental biological differences of these antigens. Although it cannot be excluded that separate functional subclasses of T-effector cells could have self-recognizers for different cell surface structures coded in I or K and D, it is more likely that the antigen parameters determine whether T cells are specific for "altered" I or "altered" K- or D-coded structures.

scholarly journals IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell–mediated rejection of systemic lymphoma in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3508-3519 ◽  
Author(s):  
John C. Markley ◽  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Tumor Rejection ◽  
Effector Memory ◽  
Transfer Model ◽  
Human T Cell

Abstract The γc-cytokines are critical regulators of immunity and possess both overlapping and distinctive functions. However, comparative studies of their pleiotropic effects on human T cell–mediated tumor rejection are lacking. In a xenogeneic adoptive transfer model, we have compared the therapeutic potency of CD19-specific human primary T cells that constitutively express interleukin-2 (IL-2), IL-7, IL-15, or IL-21. We demonstrate that each cytokine enhanced the eradication of systemic CD19+ B-cell malignancies in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice with markedly different efficacies and through singularly distinct mechanisms. IL-7– and IL-21–transduced T cells were most efficacious in vivo, although their effector functions were not as enhanced as IL-2– and IL-15–transduced T cells. IL-7 best sustained in vitro T-cell accumulation in response to repeated antigenic stimulation, but did not promote long-term T-cell persistence in vivo. Both IL-15 and IL-21 overexpression supported long-term T-cell persistence in treated mice, however, the memory T cells found 100 days after adoptive transfer were phenotypically dissimilar, resembling central memory and effector memory T cells, respectively. These results support the use of γc-cytokines in cancer immunotherapy, and establish that there exists more than 1 human T-cell memory phenotype associated with long-term tumor immunity.

Enhanced Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV Specific Central Memory T Cells by CMV Vaccine.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3014-3014
Author(s):  
Xiuli Wang ◽  
Winnie Wong ◽  
Wen-Chung Chang ◽  
Don Diamond ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Adoptive Transfer ◽  
Viral Antigen ◽  
Memory T Cells ◽  
Central Memory T Cells ◽  
Tumor Bearing ◽  
Nsg Mice

Abstract Abstract 3014 Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of cloned and bulk T cell transfer as a therapy for patients. But potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. In contrast, the adoptive transfer of viral specific T cells has shown efficient efficacy for preventing progressive viral infections and exhibited long term persistence in patients, in part due to the viral specific T cells received optimal co-stimulation after engagement of their native receptors. Conceptually, engineering CMV specific T cells with CD19CAR to provide them with a second specificity for a tumor antigen may enable the transferred T cells (bi-specific T cells) to persist or numerically expand in vivo by stimulation of the endogenous TCR by virus antigen. Moreover, bi-specific T cell can be used in treatment for B cell malignancies in allo-settings without causing GVHD due to the pre-defined non-alloreactive TCR specificity. In this study, we explored the use of CMVxCD19CAR bi-specific T cells in CD19+tumor bearing NSG mice and evaluated their antitumor activity in response to CMVpp65 antigen stimulation as a consequence of CAR transduced T cell expansion. CMV specific T cells derived from central memory T cells were selectively expanded by 2 rounds of stimulation with cGMP grade pp65 protein followed a rapid expansion containing OKT3 and feeder cells. The established CMV specific Tcm, in which majority of them are CMVpp65 tetramer positive, were then transduced with cGMP grade SIN lentivirus expressing CD19R:CD28:z/EGFRt. After stimulation with CD19 positive LCL, 40% of the resultant cells co-express pp65 tetramer and CAR as detected by EGFRt/Erbitux analysis. Functionally, the bi-specific T cells exhibit specific cytolytic activity and secret IFNg, IL2 and TNFα upon engagement with pp65 or CD19 antigen, indicating that the effector function of the bi-specific T cells can be induced through endogenous TCR or the introduced CAR. To evaluate the in vivo viral antigen driven anti-tumor efficacy of the adoptively transferred bi-specific T cells, CD19+LCL expressing GFPffluc were inoculated (i.v) into huIL-15 reconstituted NSG mice. Once the tumor engraftment was confirmed by in vivo imaging, bi-specific T cells were adoptively transferred (i.v) into the tumor bearing mice. Anti- tumor activity was observed 14 days post T cell infusion. As expected, this effect is transient and tumor re-progression occurred. In order to deliver CMV antigen for vaccine, we generated T-APC by loading CMVpp65 peptide into autologous T cells and injected the CMV T-APCs (I.v) into the bi-specific T cell treated mice, Influenza specific MP1 peptide pulsed autologous T cells were used as control T-APCs. CMV T-APC induced a second wave of antitumor activity 2 weeks post vaccine and mice survived for more than 2 months post adoptive transfer of T cells, while tumor grew vigorously when MP1-T-APCs were given as stimulators. The findings demonstrated that CD19CAR modified CMV specific T cells are capable of responding to viral antigen reactivation through their endogenous TCR, which could be used to magnify the antitumor activity of CAR transduced T cells in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 1888-1898 ◽  
Author(s):  
Xiuli Wang ◽  
Carolina Berger ◽  
ChingLam W. Wong ◽  
Stephen J. Forman ◽  
Stanley R. Riddell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Effector T Cells ◽  
Central Memory ◽  
Model Systems ◽  
Adoptive T Cell Therapy ◽  
Effector Memory ◽  
T Cell Therapy ◽  
Effector Cells

Abstract In clinical trials of adoptive T-cell therapy, the persistence of transferred cells correlates with therapeutic efficacy. However, properties of human T cells that enable their persistence in vivo are poorly understood, and model systems that enable investigation of the fate of human effector T cells (TE) have not been described. Here, we analyzed the engraftment of adoptively transferred human cytomegalovirus pp65-specific CD8+ TE cells derived from purified CD45RO+CD62L+ central memory (TCM) or CD45RO+CD62L− effector memory (TEM) precursors in an immunodeficient mouse model. The engraftment of TCM-derived effector cells (TCM/E) was dependent on human interleukin-15, and superior in magnitude and duration to TEM-derived effector cells (TEM/E). T-cell receptor Vβ analysis of persisting cells demonstrated that CD8+ TCM/E engraftment was polyclonal, suggesting that the ability to engraft is a general feature of TCM/E. CD8+ TEM/E proliferated extensively after transfer but underwent rapid apoptosis. In contrast, TCM/E were less prone to apoptosis and established a persistent reservoir of functional T cells in vivo characterized by higher CD28 expression. These studies predict that human CD8+ effector T cells derived from TCM precursors may be preferred for adoptive therapy based on superior engraftment fitness.

scholarly journals γδ T Cells from Tolerized αβ T Cell Receptor (TCR)–deficient Mice Inhibit Contact Sensitivity-Effector T Cells In Vivo, and Their Interferon-γ Production In Vitro

1996 ◽  
Vol 184 (6) ◽  
pp. 2129-2140 ◽  
Author(s):  
Marian Szczepanik ◽  
Laurel R. Anderson ◽  
Hiroko Ushio ◽  
Wlodzimierz Ptak ◽  
Michael J. Owen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Γδ T Cells ◽  
Interferon Γ ◽  
Effector T Cells ◽  
High Dose ◽  
Effector Cells

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell–mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using αβ T cell–deficient (TCRα−/−) mice, are presented here. The results clearly show that TCRα−/− mice cannot mount CS, implicating αβ T cells as the critical CS-effector cells. However, TCRα−/− mice can, after high dose tolerance, downregulate α+/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized α−/− mice are shown to include γδ TCR+ cells that also can downregulate interferon-γ production by the targeted CS-effector cells in vitro. Downregulation by γδ cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that γδ T cells cannot fulfill CS-effector functions performed by αβ T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by γδ T cells. Comparisons are likewise considered with downregulation by γδ T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.

scholarly journals Off-target effects of Cre recombinase reveal limits of adoptive T cell transfers and persistent proliferation of effector CD8 T-cells

2018 ◽  
Author(s):  
Lisa Borkner ◽  
Anja Drabig ◽  
Xiaoyan Zheng ◽  
Julia Drylewicz ◽  
Thomas Marandu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Murine Cytomegalovirus ◽  
Effector Memory ◽  
Latently Infected ◽  
The One

Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in their natural environment and observed robust long-term in vivo cycling within the TEM subset that was stronger than the one in the TCM subset. We compared the non-persistent vaccinia virus and the persisting murine Cytomegalovirus (MCMV), which induces inflationary TEM responses that remain high during viral latency. We analyzed Ki67 expression during acute, resolved and latent infection and found Ki67hiBcl2lo TEM in acutely or latently infected mice, arguing for antigen-driven TEM proliferation. In vivo labeling with deuterium showed that TEM acquired deuterium more rapidly than TCM, and were rapidly lost during chase. Similarly, antibody-mediated depletion of primed CD8 T cells in latenly infected mice revealed that TEM replenished more rapidly than TCM, suggesting that TEM cycle faster than TCM. Finally, we utilized the ability of Tamoxifen-induced Cre-ERT2 recombinase to induce chromosomal translocations when large amounts of Tamoxifen are administered for an extended time, which resulted in a selective depletion of proliferating Ki67hi cells that hardly affected the TCM subset, but drove a selective loss of Ki67hiBcl2lo effector T-cells, and an increase in the death of TEM in the spleen, arguing that TEM preferentially proliferate in the spleen. Since our results contradicted previous evidence from adoptive transfer experiments, we tested T cell homing to the spleen upon adoptive transfer. TEM homing was substantially poorer than the one of TCM, likely explaining the previously reported expansions of TCM, but not TEM, upon transfer into latently infected mice. In conclusion, our data suggest that memory inflation is largely maintained by splenic proliferation of antigen-specific TEM, rather than by continued expansion and differentiation of TCM.

scholarly journals A Novel Role for IL-6 Receptor Classic Signaling: Induction of RORγt+Foxp3+ Tregs with Enhanced Suppressive Capacity

2019 ◽  
Vol 30 (8) ◽  
pp. 1439-1453 ◽  
Author(s):  
Julia Hagenstein ◽  
Simon Melderis ◽  
Anna Nosko ◽  
Matthias T. Warkotsch ◽  
Johannes V. Richter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Th17 Cells ◽  
Inflammatory Diseases ◽  
Double Positive ◽  
T Effector Cells ◽  
Suppressive Capacity

BackgroundNew therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown.MethodsTo learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell–specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations.ResultsLack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra−/− Tregs resulted in severe aggravation of GN in mice.ConclusionsOur data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell–intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6–directed therapies for GN need to be cell-type specific.

scholarly journals CD8+ T Cell–mediated Injury In Vivo Progresses in the Absence of Effector T Cells

2001 ◽  
Vol 194 (12) ◽  
pp. 1835-1846 ◽  
Author(s):  
Barbara A. Small ◽  
Sarah A. Dressel ◽  
Christopher W. Lawrence ◽  
Donald R. Drake ◽  
Mark H. Stoler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tissue Injury ◽  
Cd8 T Cell ◽  
Effector T Cells ◽  
Type Ii ◽  
Pulmonary Injury ◽  
Alveolar Cells ◽  
Type Ii Alveolar Cells

Tissue injury is a common sequela of acute virus infection localized to a specific organ such as the lung. Tissue injury is an immediate consequence of infection with lytic viruses. It can also result from the direct destruction of infected cells by effector CD8+ T lymphocytes and indirectly through the action of the T cell–derived proinflammatory cytokines and recruited inflammatory cells on infected and uninfected tissue. We have examined CD8+ T cell–mediated pulmonary injury in a transgenic model in which adoptively transferred, virus-specific cytotoxic T lymphocytes (CTLs) produce lethal, progressive pulmonary injury in recipient mice expressing the viral target transgene exclusively in the lungs. We have found that over the 4–5 day course of the development of lethal pulmonary injury, the effector CTLs, while necessary for the induction of injury, are present only transiently (24–48 h) in the lung. We provide evidence that the target of the antiviral CD8+ T cells, the transgene expressing type II alveolar cells, are not immediately destroyed by the effector T cells. Rather, after T cell–target interaction, the type II alveolar cells are stimulated to produce the chemokine monocyte chemoattractant protein 1. These results reinforce the concept that, in vivo, the cellular targets of specific CTLs may participate directly in the development of progressive tissue injury by activating in response to interaction with the T cells and producing proinflammatory mediators without sustained in vivo activation of CD8+ T cell effectors.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

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