Miles, John J.

Abstract Multiple myeloma (MM) is a largely incurable hematological malignancy characterized by clonal expansion of plasma cells in bone marrow (BM). Autologous stem cell transplantation (ASCT) remains a standard treatment for MM, allows intensive cytoreduction and induces inflammation in the context of lymphodepletion. We hypothesized that SCT may overcome myeloma-induced immune defects to restore immune-equilibrium. To address this, we developed a model of ASCT for MM using Vk*MYC myeloma. Surprisingly, we demonstrate the induction of T cell-dependent MM control after SCT as recipients of syngeneic BM grafts containing T cells had prolonged survival and reduced tumor burden compared to BM alone. This anti-myeloma activity was independent of γδ T, natural killer (NK) and NK T cells and was dependent on CD4+and CD8+ T cells. MM-primed T cells, harvested from SCT recipients with controlled MM, protected against relapse in secondary recipients and were Vk*MYC clone-specific. Recipients of MM-primed T cells had increased central memory CD8+ T cells and transfer of MM-primed CD8+ T cells was sufficient to prevent relapse. Furthermore, TCR sequencing of CD8+ T cells from mice with controlled MM revealed a higher clonotype overlap relative to MM-free SCT recipients, consistent with MM-specific T cells. Donor IFNγ secretion and signaling was critical to protective immunity and CD8+ T cells from mice with relapsed MM had impaired IFNγ production compared to those with controlled disease. Importantly, IFNγ production was profoundly augmented by CD137 agonist treatment such that majority of mice controlled disease. These data provide new insights into the mechanisms of action of SCT in myeloma and suggest the use of CD137 agonists to prevent relapse.

Physical activity (PA) is inversely related with body fat, insulin resistance (IR), and leptin. However, these associations are not clear in patients after gastric bypass surgery (GBS). We hypothesized an inverse relation between PA, BMI, IR, and leptin; and a positive relation between leptin, BMI, and IR in a group of 9 patients after GBS, 8 obese (Ob), and 7 non‐obese (N‐Ob) controls (age=39.0±5.7, 27.7±6.9, 28.0±10.2 yr, respectively). Subjects wore an accelerometer from 6‐7 days, and underwent measurements of height, weight, DEXA, blood glucose, insulin and leptin. IR was estimated using HOMA. Between group differences in PA, BMI, IR, and leptin were determined with ANOVA; and the relation between these variables was determined with simple linear regressions. Between group differences were observed for BMI (Ob=38.7±1.6, GBS=30.4±1.5, N‐Ob=23.8±1.7 kg/m2), IR (Ob=3.0±0.5, GBS=1.1±0.5, N‐Ob=0.7±0.5), and leptin (Ob=55.9, GBS=28.7±4.5, N‐Ob=5.1±5.6 ng/ml). PA was not different between groups. Inverse associations were observed between PA and BMI (r= ‐ 0.55), and leptin (r= ‐ 0.56); and positive associations were observed between leptin and BMI (r= 0.92), and IR (r= 0.63). PA appears to be associated with the control of obesity and IR possibly via reductions in leptin, a mechanism that requires further study. Funded by NIH CTSA UL1‐RR24150, NIH CTSA KL2‐ RR024151

The thermic effect of food (TEF) is partially mediated by β‐adrenergic receptors (βAR) and is an important component of daily energy expenditure. Greater sympathetic activity has been associated with decreased BAR responsiveness. We hypothesized that muscle sympathetic nerve activity (MSNA) would be inversely correlated with the βAR component of TEF (TEF β ). We measured MSNA (microneurography) and energy expenditure (EE) (indirect calorimetry) before and for 5 h after a mixed meal (40% daily EE). Measurements were made during systemic βAR blockade with propranolol (BB) and saline (NS) infusion on separate days in 10 healthy subjects (2 female, age 28±2 y, BMI 24.4±0.6 kg/m 2 ). Resting MSNA was 39±3 bursts/100 heartbeats (hb). Within 1 h after the meal, MSNA significantly increased during both NS (12±3 bursts/100 hb) and BB (18±4) but was not significantly different between the two days, (P=0.375). The maximum increase in REE after the meal was 269±29 (NS) and 198±22 (BB) kcal/24 h (P<0.05). TEF β (TEF β = AUC TEF NS – AUC TEF BB ) was 48±20% of total TEF. TEF β was positively correlated with resting MSNA (R 2 =0.70, P<0.05). Contrary to our hypothesis, we found that greater levels of MSNA were associated with greater TEF β . These results indicated that resting MSNA may be an important contributor to daily energy expenditure. Studies are needed to determine if greater MSNA is associated with a resistance to weight gain. The project described was supported by the Mayo Foundation for Medical Education and Research and NIH Grant Numbers K23 DK82424, P30 DK50456, and UL1 RR024150.

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease. Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response. Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.

Abstract Context A better understanding of nocturnal regulation of glucose homeostasis will provide the framework for designing rational therapeutic strategies to improve the management of overnight glucose in patients with type 2 diabetes (T2D). Objective To establish the nocturnal pattern and regulation of glucose production (EGP) in humans and to determine whether the pattern is dysregulated in people with T2D. Design Subjects were infused with [3-3H] glucose overnight. Arterial blood samples were drawn for hormones and analytes to estimate EGP throughout the night. Deuterium-labeled water was provided to measure gluconeogenesis (GNG) using the hexamethylenetetramine method of Landau. Setting Mayo Clinic Clinical Research Trials Unit, Rochester, MN, USA. Participants and Interventions A total of 43 subjects [23 subjects with T2D and 20 nondiabetic (ND) subjects comparable for age and body mass index] were included in this study. Main Outcome(s) Measure(s) Glucose and EGP. Results Plasma glucose, C-peptide, and glucagon concentrations were higher throughout the night, whereas insulin concentrations were higher in subjects with T2D vs ND subjects at 1:00 and 4:00 am but similar at 7:00 am. EGP was higher in the subjects with T2D than in the ND subjects throughout the night (P &lt; 0.001). Glycogenolysis (GGL) fell and GNG rose, resulting in significantly higher (P &lt; 0.001) rates of GNG at 4:00 and 7:00 am and significantly (P &lt; 0.001) higher rates of GGL at 1:00, 4:00, and 7:00 am in T2D as compared with ND. Conclusions These data imply that optimal therapies for T2D for nocturnal/fasting glucose control should target not only the absolute rates of EGP but also the contributing pathways of GGL and GNG sequentially.

To determine whether the sensitivity of adipose tissue lipolysis to catecholamines is increased in poorly controlled insulin-dependent diabetes, the lipolytic response to epinephrine was measured in seven nondiabetic volunteers and seven poorly controlled diabetic subjects with use of [1-(14)C]palmitate as a tracer. Subjects received sequential 1-h infusions of epinephrine, which produced epinephrine concentrations of approximately 1,000, approximately 1,750, approximately 3,500, and approximately 6,000 pmol/l. A pancreatic clamp was used to maintain constant plasma hormone levels. Concentration-response curves were constructed for each subject from the integrated lipolytic response during each epinephrine infusion. There was no difference in maximal lipolytic response (117 +/- 19 vs. 152 +/- 11 mumol.kg-1.h-1) or in maximally effective (3,171 +/- 267 vs. 3,357 +/- 349 pmol/l) or half-maximally effective (1,081 +/- 109 vs. 1,015 +/- 120 pmol/l) epinephrine concentrations between nondiabetic and diabetic subjects, respectively (all P = NS). In control subjects, maximum beta-hydroxybutyrate concentrations were achieved at lower epinephrine concentrations than those required for a maximum lipolytic effect. Thus, under pancreatic clamp conditions, the lipolytic response to epinephrine in nondiabetic and diabetic subjects was similar.

An assay for plasma lipoprotein lipase activity was used without prior injection of heparin to study arteriovenous differences of lipases across skeletal muscle and adipose tissue of normal male volunteers. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities and triglyceride?concentrations were measured in arterial plasma and in venous effluent plasma from forearm skeletal muscle and subcutaneous abdominal adipose tissue, in the postabsorptive state and after a mixed meal. Triglyceride clearance by the tissues was greater across adipose tissue than across muscle. There were no arteriovenous differences for HTGL activity. In the postabsorptive state skeletal muscle released LPL activity, but adipose tissue did not. Postprandially the arterial LPL and HTGL activities did not change. LPL activity in adipose tissue venous effluent rose, whereas that in muscle venous effluent decreased. These results show that the release of LPL from subcutaneous adipose and forearm tissues is regulated differently, reflecting in vivo differences in LPL regulation at the tissue level.

Controversy exists regarding whether plasma glucose concentrations are independently involved in the regulation of adipose tissue lipolysis. In the present study, six subjects with insulin-dependent diabetes and six nondiabetic volunteers were studied during infusion of somatostatin, growth hormone, and insulin at rates designed to maintain basal rates of lipolysis, which was traced using a constant infusion of [1-14C]palmitate. A euglycemic (approximately 5 mmol/l) clamp was performed for 3 h, followed by 3 h of hyperglycemia (approximately 9 and approximately 11 mmol/l in nondiabetic and diabetic subjects, respectively). Ten nondiabetic subjects were studied during 6 h of euglycemia to exclude time-dependent changes in lipolysis. The results showed that palmitate concentrations did not change between euglycemia and hyperglycemia in either group [118 +/- 10 vs. 132 +/- 14 mumol/l and 145 +/- 21 vs. 134 +/- 15 mumol/l in nondiabetic and diabetic subjects, respectively; both P = not significant (NS)]. Similarly, palmitate rate of appearance (Ra) did not change during hyperglycemia (1.0 +/- 0.1 and 1.7 +/- 0.4 mumol.kg-1.min-1 in nondiabetic and diabetic subjects, respectively) compared with euglycemia (1.0 +/- 0.1 and 1.6 +/- 0.4 mumol.kg-1.min-1 in nondiabetic and diabetic subjects, respectively; P = NS). Palmitate concentrations and Ra did not change during 6 h of euglycemia in nondiabetic volunteers. Thus hyperglycemia per se has no effect on free fatty acid turnover. Changes in lipolysis that occur coincident with hyperglycemia are probably due to changes in other circulating substrates or hormones known to affect lipolysis.