Clinical Utility of the OmniGraf Biomarker Panel in the Care of Kidney Transplant Recipients (CLARITY): Protocol for a Prospective, Multisite Observational Study.

BACKGROUND: Death with a functioning allograft has become the leading category of graft loss in kidney transplant recipients at all time points. Previous analyses have demonstrated that causes of death in kidney transplant recipients are predominated by comorbidities strongly associated with immunosuppressant medications. Adverse drug events (ADEs) have been strongly associated with nonadherence, health care utilization, and graft loss; clinicians face a difficult decision on whether making immunosuppressant adjustments in the face of ADEs will improve symptomology or simply increase the risk of acute rejection. Clinicians also face a treatment quandary in 50% of kidney transplant recipients with stage 3 or worse chronic kidney disease at 1 year post transplantation, as progressive decline in renal function has been strongly associated with inferior allograft survival. OBJECTIVE: The primary objective of the CLinical Utility of the omnigrAf biomarkeR Panel In The Care of kidneY Transplant Recipients (CLARITY) trial is to evaluate change in renal function over time in kidney transplant recipients who are undergoing OmniGraf monitoring in conjunction with monitoring of their medication-related symptom burden (MRSB). A secondary objective of this study is to identify the impact of OmniGraf use in conjunction with patient-reported MRSB as part of clinical care on patients' self-efficacy and quality of life. METHODS: CLARITY is a 3-year prospective, multisite, observational study of 2000 participants with a matched control, measuring the impact of real-time patients' MRSB and the OmniGraf biomarker panel on change in renal function over time. Secondary outcome measures include the Patient-Reported Outcomes Measurement Information System (PROMIS) Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a; the PROMIS-29 Profile (version 2.1); the PROMIS Depression Scale, hospitalizations-subcategorized for hospitalizations owing to infections; treated rejections, MRSB, and proportion of participants with overall graft survival at year 3 post transplantation; graft loss or death during the 3-year study follow-up period; and change in provider satisfaction. RESULTS: The primary outcome measure of the study will be a comparison of the slope change in estimated glomerular filtration rate from baseline to the end of follow-up between study participants and a matched control group. Secondary outcome measures include changes over time in PROMIS Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a, the PROMIS-29 Profile (version 2.1), and PROMIS Depression Scale in the study group, as well as a comparison of hospitalizations and causes, rejections, and graft and patient survival compared between participants and a matched cohort. The anticipated first enrollment in the study is October 2022 with data analysis and publication expected in October 2027. CONCLUSIONS: Through this report, we describe the study design, methods, and outcome measures that will be utilized in the ongoing CLARITY trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT05482100; https://clinicaltrials.gov/ct2/show/NCT05482100. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/41020.

mTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells.

In myeloid dendritic cells (DC), deletion of the mechanistic target of rapamycin complex 2 (TORC2) results in an augmented pro-inflammatory phenotype and T cell stimulatory activity; however, the underlying mechanism has not been resolved. Here, we demonstrate that mouse bone marrow-derived TORC2-deficient myeloid DC (TORC2-/- DC) utilize an altered metabolic program, characterized by enhanced baseline glycolytic function compared to wild-type WT control (Ctrl) DC, increased dependence on glycolytic ATP production, elevated lipid content and higher viability following stimulation with LPS. In addition, TORC2-/- DC display an increased spare respiratory capacity (SRC) compared to WT Ctrl DC; this metabolic phenotype corresponds with increased mitochondrial mass and mean mitochondrial DNA copy number, and failure of TORC2-/- DC mitochondria to depolarize following LPS stimulation. Our data suggest that the enhanced metabolic activity of TORC2-/- DC may be due to compensatory TORC1 pathway activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1(JAK1), and the activation of downstream targets of TORC1, including p70S6K, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and CD36 (fatty acid translocase). These enhanced TORC1 pathway activities may culminate in increased expression of the nuclear receptor peroxisome proliferator-activated receptor γ (Pparγ) that regulates fatty acid storage, and the transcription factor sterol regulatory element-binding transcription factor 1 (Srebf1). Taken together, our data suggest that TORC2 may function to restrain TORC1-driven metabolic activity and mitochondrial regulation in myeloid DC.

Determining a Resting Metabolic Rate Prediction Equation for Collegiate Female Athletes.

Watson, AD, Zabriskie, HA, Witherbee, KE, Sulavik, A, Gieske, BT, and Kerksick, CM. Determining a resting metabolic rate prediction equation for collegiate female athletes. J Strength Cond Res 33(9): 2426-2432, 2019-A lack of evidence exists regarding the accuracy of common resting metabolic rate (RMR) prediction equations in athletic female populations. The purpose of this research was to measure RMR in a large cohort of NCAA Division II female athletes and use regression techniques to develop new prediction equations. Sixty-six female athletes from 11 different sports completed this protocol, which included skinfold measurements followed by an RMR assessment using indirect calorimetry. The average RMR was 1,466 ± 150 kcal·d. Many between-sport differences in body composition were identified, with gymnastics athletes having the lowest body fat percentage (p < 0.05) and basketball athletes having the greatest absolute fat-free mass (p < 0.05). Resting metabolic rate was moderately correlated (p < 0.05) with height (r = 0.52), total mass (r = 0.59), and fat-free mass (r = 0.54). Two equations were developed, both of which were more accurate for this population than other RMR prediction equations. One of the new equations, which used height and body mass as covariates (equation 1), was slightly more accurate than the equation using body composition parameters (equation 2). The new equations were cross-validated using a randomly selected subset (n = 22) of the original sample. The subset did not show statistically different results from the remainder of the sample (n = 44) between equation 1 (p = 0.083) and equation 2 (p = 0.22). Equation 1, which had more easily measurable parameters, exhibited heightened accuracy, which has important implications for implementation among athletes, coaches, and athletic support staff.

mTORC2 deficiency in cutaneous dendritic cells potentiates CD8+ effector T cell responses and accelerates skin graft rejection.

Mechanistic target of rapamycin (mTOR) complex (mTORC)1 and mTORC2 regulate the differentiation and function of immune cells. While inhibition of mTORC1 antagonizes dendritic cell (DC) differentiation and suppresses graft rejection, the role of mTORC2 in DCs in determining host responses to transplanted tissue remains undefined. Using a mouse model in which mTORC2 was deleted specifically in CD11c+ DCs (TORC2DC-/- ), we show that the transplant of minor histocompatibility Ag (HY)-mismatched skin grafts from TORC2DC-/- donors into wild-type recipients results in accelerated rejection characterized by enhanced CD8+ T cell responses in the graft and regional lymphoid tissue [Correction added on January 9, 2019, after first online publication: in the previous sentence, major was changed to minor]. Similar enhancement of CD8+ effector T cell responses was observed in MHC-mismatched recipients of TORC2DC-/- grafts. Augmented CD8+ T cell responses were also observed in a delayed-type hypersensitivity model in which mTORC2 was absent in cutaneous DCs. These elevated responses could be ascribed to an increased T cell stimulatory phenotype of TORC2DC-/- and not to enhanced lymph node homing of the cells. In contrast, rejection of ovalbumin transgenic skin grafts in TORC2DC-/- recipients was unaffected. These findings suggest that mTORC2 in skin DCs restrains effector CD8+ T cell responses and have implications for understanding of the influence of mTOR inhibitors that target mTORC2 in transplant.

Rictor deficiency in dendritic cells exacerbates acute kidney injury.

Dendritic cells (DCs) are critical initiators of innate immunity in the kidney and orchestrate inflammation following ischemia-reperfusion injury. The role of the mammalian/mechanistic target of rapamycin (mTOR) in the pathophysiology of renal ischemia-reperfusion injury has been characterized. However, the influence of DC-based alterations in mTOR signaling is unknown. To address this, bone marrow-derived mTORC2-deficient (Rictor-/-) DCs underwent hypoxia-reoxygenation and then analysis by flow cytometry. Adoptive transfer of wild-type or Rictor-/- DC to C57BL/6 mice followed by unilateral or bilateral renal ischemia-reperfusion injury (20 min ischemia) was used to assess their in vivo migratory capacity and influence on tissue injury. Age-matched male DC-specific Rictor-/- mice or littermate controls underwent bilateral renal ischemia-reperfusion, followed by assessment of renal function, histopathology, and biomolecular and cell infiltration analysis. Rictor-/- DCs expressed more costimulatory CD80/CD86 but less coinhibitory programmed death ligand 1 (PDL1), a pattern that was enhanced by hypoxia-reoxygenation. They also demonstrated enhanced migration to the injured kidney and induced greater tissue damage. Following ischemia-reperfusion, Rictor-/- DC mice developed higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production compared to littermate controls. Additionally, a greater influx of both neutrophils and T cells was seen in Rictor-/- DC mice, along with CD11c+MHCII+CD11bhiF4/80+ renal DC, that expressed more CD86 but less PDL1. Thus, DC-targeted elimination of Rictor enhances inflammation and migratory responses to the injured kidney, highlighting the regulatory roles of both DCs and Rictor in the pathophysiology of acute kidney injury.

Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection.

BACKGROUND: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. METHODS: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. RESULTS: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. CONCLUSIONS: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.

Intratumoral delivery of mTORC2-deficient dendritic cells inhibits B16 melanoma growth by promoting CD8(+) effector T cell responses.

Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses. In cancer, DC-based vaccines have proven to be safe and to elicit protective and therapeutic immunological responses. Recently, we showed that specific mTORC2 (mechanistic target of rapamycin complex 2) deficiency in DC enhances their ability to promote Th1 and Th17 responses after LPS stimulation. In the present study, bone marrow-derived mTORC2-deficient (Rictor(-/-)) DC were evaluated as a therapeutic modality in the murine B16 melanoma model. Consistent with their pro-inflammatory profile (enhanced IL-12p70 production and low PD-L1 expression versus control DC), intratumoral (i.t.) injection of LPS-activated Rictor(-/-) DC slowed B16 melanoma growth markedly in WT C57BL/6 recipient mice. This antitumor effect was abrogated when Rictor(-/-) DC were injected i.t. into B16-bearing Rag(-/-) mice, and also after selective CD8(+) T cell depletion in wild-type hosts in vivo, indicating that CD8(+) T cells were the principal regulators of tumor growth after Rictor(-/-) DC injection. I.t. administration of Rictor(-/-) DC also reduced the frequency of myeloid-derived suppressor cells within tumors, and enhanced numbers of IFNγ(+) and granzyme-B(+) cytotoxic CD8(+) T cells both in the spleens and tumors of treated animals. These data suggest that selective inhibition of mTORC2 activity in activated DC augments their pro-inflammatory and T cell stimulatory profile, in association with their enhanced capacity to promote protective CD8(+) T cell responses in vivo, leading to slowed B16 melanoma progression. These novel findings may contribute to the design of more effective DC-based vaccines for cancer immunotherapy.

mTORC2 Deficiency in Myeloid Dendritic Cells Enhances Their Allogeneic Th1 and Th17 Stimulatory Ability after TLR4 Ligation In Vitro and In Vivo.

The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. In this study, to our knowledge, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow-derived DC from conditional Rictor(-/-) mice exhibit lower coinhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNF-α, IL-12p70, and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor(-/-) DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ(+) and IL-17(+), but not IL-10(+) or CD4(+)Foxp3(+) regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor(-/-) DC. Using novel CD11c-specific Rictor(-/-) mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that proinflammatory cytokines produced by Rictor(-/-) DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC proinflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation.

Assembly of human organs from stem cells to study liver disease.

Recently, significant developments in the field of liver tissue engineering have raised new possibilities for the study of complex physiological and pathophysiological processes in vitro, as well as the potential to assemble entire organs for transplantation. Human-induced pluripotent stem cells have been differentiated into relatively functional populations of hepatic cells, and novel techniques to generate whole organ acellular three-dimensional scaffolds have been developed. In this review, we highlight the most recent advances in organ assembly regarding the development of liver tissue in vitro. We emphasize applications that involve multiple types of cells with a biomimetic spatial organization for which three-dimensional configurations could be used for drug development or to explain mechanisms of disease. We also discuss applications of liver organotypic surrogates and the challenges of translating the highly promising new field of tissue engineering into a proven platform for predicting drug metabolism and toxicity.