Matthew P. Smeltzer

Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

R2* magnetic resonance imaging of the liver in patients with iron overload

R2* magnetic resonance imaging (R2*-MRI) can quantify hepatic iron content (HIC) by noninvasive means but is not fully investigated. Patients with iron overload completed 1.5T R2*-MRI examination and liver biopsy within 30 days. Forty-three patients (sickle cell anemia, n = 32; beta-thalassemia major, n = 6; and bone marrow failure, n = 5) were analyzed: median age, 14 years, median transfusion duration, 15 months, average (+/-SD) serum ferritin 2718 plus or minus 1994 ng/mL, and average HIC 10.9 plus or minus 6.8 mg Fe/g dry weight liver. Regions of interest were drawn and analyzed by 3 independent reviewers with excellent agreement of their measurements (intraclass correlation coefficient = 0.98). Ferritin and R2*-MRI were weakly but significantly associated (range of correlation coefficients among the 3 reviewers, 0.41-0.48; all P < .01). R2*-MRI was strongly associated with HIC for all 3 reviewers (correlation coefficients, 0.96-0.98; all P < .001). This high correlation confirms prior reports, calibrates R2*-MRI measurements, and suggests its clinical utility for predicting HIC using R2*-MRI. This study was registered at www.clinicaltrials.gov as #NCT00675038.

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

Obesity in Survivors of Childhood Acute Lymphoblastic Leukemia and Lymphoma

PURPOSE We evaluated the long-term effects of treatment on the body mass index (BMI) of children with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma who received one of three CNS-directed therapies: intrathecal methotrexate with intravenous high-dose methotrexate (1 g/m2), intrathecal methotrexate with 18 Gy cranial radiation, or intrathecal methotrexate with 24 Gy cranial radiation. PATIENTS AND METHODS Between 1979 and 1984, 456 children with newly diagnosed ALL and lymphoma were enrolled onto a single protocol at St Jude Childrens Research Hospital (Memphis, TN). The heights and weights of 422 of the children were measured at diagnosis, during treatment, at the end of therapy, and approximately every 6 to 12 months thereafter. Patients who had attained their adult height at the time of analysis (n = 248) were placed in weight categories based on their BMI, BMI percentile, or weight-for-length percentile depending on age. RESULTS The overall percentage of survivors who were overweight or obese approximated rates prevalent in the general population of the United States. Young age (< 6 years) and overweight/obesity at diagnosis were the best predictors of obesity at adult height. The rate of BMI increase did not differ significantly between children who received radiation and those who did not, nor between patients who received 18 or 24 Gy of cranial radiation. CONCLUSION BMI weight category at diagnosis, rather than type of CNS treatment received, predicted adult weight in long-term survivors of childhood hematologic malignancies.

Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia.

Hydroxyurea therapy has proven laboratory and clinical efficacies for children with sickle cell anemia (SCA). When administered at maximum tolerated dose (MTD), hydroxyurea increases fetal hemoglobin (HbF) to levels ranging from 10% to 40%. However, interpatient variability of percentage of HbF (%HbF) response is high, MTD itself is variable, and accurate predictors of hydroxyurea responses do not currently exist. HUSTLE (NCT00305175) was designed to provide first-dose pharmacokinetics (PK) data for children with SCA initiating hydroxyurea therapy, to investigate pharmacodynamics (PD) parameters, including HbF response and MTD after standardized dose escalation, and to evaluate pharmacogenetics influences on PK and PD parameters. For 87 children with first-dose PK studies, substantial interpatient variability was observed, plus a novel oral absorption phenotype (rapid or slow) that influenced serum hydroxyurea levels and total hydroxyurea exposure. PD responses in 174 subjects were robust and similar to previous cohorts; %HbF at MTD was best predicted by 5 variables, including baseline %HbF, whereas MTD was best predicted by 5 variables, including serum creatinine. Pharmacogenetics analysis showed single nucleotide polymorphisms influencing baseline %HbF, including 5 within BCL11A, but none influencing MTD %HbF or dose. Accurate prediction of hydroxyurea treatment responses for SCA remains a worthy but elusive goal.

On the Pathogenicity of Autoantigen-Specific T Cell Receptors

OBJECTIVE—Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing β-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo. RESEARCH DESIGN AND METHODS—We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2β/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared. RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2β/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42–56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice. CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and β-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

Evaluation of a comprehensive transcranial doppler screening program for children with sickle cell anemia.

Although transcranial Doppler ultrasonography (TCD) screening is effective in identifying children with sickle cell anemia (SCA) who are at high risk of stroke, not all children are screened. In 2003, we instituted a comprehensive TCD screening program designed to screen all at‐risk children treated at our sickle cell center.

The natural history of conditional transcranial Doppler flow velocities in children with sickle cell anaemia.

Children with sickle cell anaemia (SCA) and conditional transcranial Doppler (TCD) [time‐averaged mean velocity (TAMV) 170–199 cm/s] have increased risk of primary stroke, but receive no specific therapy. Some will convert to abnormal velocities (TAMV ≥200 cm/s) with further increase in stroke risk. In 2003, our centre initiated universal TCD screening, targeting all children (aged 2–16 years) with SCA. TCD examinations were repeated at intervals based on initial results. To determine rates and risk factors for TCD conversion, we reviewed all examinations since 2003, excluding patients receiving hydroxycarbamide (hydroxyurea) or transfusions. Of the eligible population, 274 children (98%) were screened at a median age of 7·1 years (range 1·7–18·2). Fifty‐four patients (20%) had conditional TAMV either on initial screening or a subsequent examination. The 18‐month cumulative incidence of conversion from conditional to abnormal TAMV was 23%. Age, initial TAMV, laboratory values, blood pressure and oxygen saturation were not significantly associated with conversion. Our cohort provides systematic longitudinal evaluation of an unselected paediatric population universally screened and retested at regular intervals. Our data document a high conversion rate to abnormal velocities among untreated children with SCA. Therapy should be considered for the prevention of conversion to abnormal TCD velocities.

HYDROXYUREA TREATMENT DECREASES GLOMERULAR HYPERFILTRATION IN CHILDREN WITH SICKLE CELL ANEMIA

Glomerular hyperfiltration and microalbuminuria/proteinuria are early manifestations of sickle nephropathy. The effects of hydroxyurea therapy on these renal manifestations of sickle cell anemia (SCA) are not well defined. Our objective was to investigate the effects of hydroxyurea on glomerular filtration rate (GFR) measured by 99mTc‐DTPA clearance, and on microalbuminuria/proteinuria in children with SCA. Hydroxyurea study of long‐term effects (HUSTLE) is a prospective study (NCT00305175) with the goal of describing the long‐term cellular, molecular, and clinical effects of hydroxyurea therapy in SCA. Glomerular filtration rate, urine microalbumin, and serum cystatin C were measured before initiating hydroxyurea therapy and then repeated after 3 years. Baseline and Year 3 values for HUSTLE subjects were compared using the Wilcoxon Signed Rank test. Associations between continuous variables were evaluated using Spearman correlation coefficient. Twenty‐three children with SCA (median age 7.5 years, range, 2.5–14.0 years) received hydroxyurea at maximum tolerated dose (MTD, 24.4 ± 4.5 mg/kg/day, range, 15.3–30.6 mg/kg/day). After 3 years of treatment, GFR measured by 99mTc‐DTPA decreased significantly from 167 ± 46 mL/min/1.73 m2 to 145 ± 27 mL/min/1.73 m2 (P = 0.016). This decrease in GFR was significantly associated with increase in fetal hemoglobin (P = 0.042) and decrease in lactate dehydrogenase levels (P = 0.035). Urine microalbumin and cystatin C levels did not change significantly. Hydroxyurea at MTD is associated with a decrease in hyperfiltration in young children with SCA. Am. J. Hematol., 88:116–119, 2013.

Assessment of genotoxicity associated with hydroxyurea therapy in children with sickle cell anemia.

Hydroxyurea induces fetal hemoglobin, improves laboratory parameters, and ameliorates clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety in this patient population remain incompletely defined. Although generally considered non-DNA reactive, an important safety concern is that hydroxyurea may indirectly cause genotoxic damage. To better address this safety issue of hydroxyurea in patients with SCA, we measured the production of micronuclei (MN) in red blood cells (RBCs) as a marker of genotoxicity. Blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (ClinicalTrials.gov NCT00305175). Flow cytometry quantified circulating MN-containing erythrocyte sub-populations before and during hydroxyurea exposure. The frequency of micronucleated reticulocytes (MN-CD71(+)) and micronucleated mature erythrocytes (MN-RBC) was then tested for associations with laboratory and clinical data. In cross-sectional analysis of 293 blood samples from 105 children with SCA and a median of 2 years of hydroxyurea therapy, exposure to hydroxyurea was associated with significantly increased frequencies of MN-CD71(+) and MN-RBC compared to baseline. The increases were evident by 3 months of therapy, and did not escalate further with up to 12 years of continuous drug exposure. In prospective longitudinal analysis, substantial inter-individual variation in the effect of hydroxyurea on %MN-CD71(+) was observed that was associated with the expected laboratory effects of hydroxyurea. In conclusion, clinically relevant exposure to hydroxyurea is associated with increased MN production consistent with erythroblast genotoxicity but with substantial inter-patient variability. Associations between increased %MN-CD71(+) and laboratory benefits suggest that hydroxyurea effects on MN production may be related to individual patient sensitivity to hydroxyurea within the bone marrow.