Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.

Pharmacology of Chimeric Antigen Receptor-Modified T Cells.

Cell-based immunotherapies using T cells that are engineered to express a chimeric antigen receptor (CAR-T cells) are an effective treatment option for several B cell malignancies. Compared with most drugs, CAR-T cell products are highly complex, as each cell product is composed of a heterogeneous mixture of millions of cells. The biodistribution and kinetics of CAR-T cells, following administration, are unique given the ability of T cells to actively migrate as well as replicate within the patient. CAR-T cell therapies also have multiple mechanisms of action that contribute to both their antitumor activity and their toxicity. This review provides an overview of the unique pharmacology of CAR-T cells, with a focus on CD19-targeting and B cell maturation antigen (BCMA)-targeting CAR-T cells.

F77 antigen is a promising target for adoptive T cell therapy of prostate cancer.

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has made significant success in treating hematological malignancies, paving the way for solid tumors like prostate cancer. However, progress is impeded by a paucity of suitable target antigens. A novel carbohydrate antigen, F77, is expressed on both androgen-dependent and androgen-independent prostate cancer cells, making it a potential immunotherapy target. This study entails the generation and evaluation of a second-generation CAR against a carbohydrate antigen on malignant prostate cancer cells. Using a single chain fragment variable (scFv) from an F77-specific mouse monoclonal antibody, we created second-generation CARs with CD28 and CD137 (4-1BB) costimulatory signals. F77 expressing lentiviral CAR T cells produce cytokines and kill tumor cells in a F77 expression-dependent manner. These F77-specific CAR T cells eradicate prostate tumors in a human xenograft model employing PC3 cells. These findings validate F77 as a promising immunotherapeutic target for prostate cancer and other malignancies with this aberrant carbohydrate structure.

Enhancing CAR T function with the engineered secretion of C. perfringens neuraminidase.

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Debulking SARS-CoV-2 in saliva using angiotensin converting enzyme 2 in chewing gum to decrease oral virus transmission and infection.

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others.

Evaluation of a Nanoparticle-Based Busulfan Immunoassay for Rapid Analysis on Routine Clinical Analyzers.

BACKGROUND: Busulfan is an alkylating agent used in allogeneic hematopoietic stem cell transplantation for various malignant and nonmalignant disorders. Therapeutic drug monitoring of busulfan is common because busulfan exposure has been linked to veno-occlusive disease, disease relapse, and failed engraftment. The authors developed an automated immunoassay, along with stable calibrators and controls, and quantified busulfan in sodium heparin plasma. METHODS: The authors evaluated a homogenous nanoparticle immunoassay, the MyCare Oncology Busulfan Assay Kit (Saladax Biomedical, Inc), for precision, sensitivity, accuracy, and linearity on an open channel clinical chemistry analyzer; they compared the method with 2 mass spectrometry methods (liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry), using anonymized, remnant patient samples. RESULTS: The coefficients of variation for repeatability and within-laboratory precision were ≤9.0%. The linear range was 150-2000 ng/mL; samples up to 6000 ng/mL can be measured with sample dilution. Measured values deviated by ≤14% from assigned values. Comparison between validated mass spectrometry methods resulted in a correlation coefficient R ≥ 0.995. CONCLUSIONS: The MyCare Busulfan Assay Kit shows the precision, accuracy, linearity, and test range for performing busulfan concentration measurements in sodium heparin plasma on routine clinical chemistry analyzers.

Ligand-Induced Degradation of a CAR Permits Reversible Remote Control of CAR T Cell Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR)-modified T cells are endowed with novel antigen specificity and are most often administered to patients without an engineered mechanism to control the CAR T cells once infused. "Suicide switches" such as the small molecule-controlled, inducible caspase-9 (iCas9) system afford the ability to selectively eliminate engineered T cells; however, these approaches are designed for all-or-none, irreversible termination of an ongoing immune response. In order to permit reversible and adjustable modulation, we have created a CAR that is capable of on-demand downregulation by fusing the CAR to a previously developed ligand-induced degradation (LID) domain. Addition of a small molecule ligand triggers exposure of a cryptic degron within the LID domain, resulting in proteasomal degradation of the CAR-LID fusion protein and loss of CAR on the surface of T cells. This fusion construct allowed for reversible and "tunable" inhibition of CAR T cell activity in vitro. Delivery of the triggering molecule in CAR-LID-treated tumor-bearing mice temporarily reduced CAR activity through modulation of CAR surface expression. The ability to more flexibly modulate CAR T cell expression through a small molecule provides a platform for controlling possible adverse side effects, as well as preclinical investigations of CAR T cell biology.

Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene.

Cell-based therapeutics have considerable promise across diverse medical specialties; however, reliable human imaging of the distribution and trafficking of genetically engineered cells remains a challenge. We developed positron emission tomography (PET) probes based on the small-molecule antibiotic trimethoprim (TMP) that can be used to image the expression of the Escherichia coli dihydrofolate reductase enzyme (eDHFR) and tested the ability of [18F]-TMP, a fluorine-18 probe, to image primary human chimeric antigen receptor (CAR) T cells expressing the PET reporter gene eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc). Engineered T cells showed an approximately 50-fold increased bioluminescent imaging signal and 10-fold increased [18F]-TMP uptake compared to controls in vitro. eDHFR-expressing anti-GD2 CAR T cells were then injected into mice bearing control GD2- and GD2+ tumors. PET/computed tomography (CT) images acquired on days 7 and 13 demonstrated early residency of CAR T cells in the spleen followed by on-target redistribution to the GD2+ tumors. This was corroborated by autoradiography and anti-human CD8 immunohistochemistry. We found a high sensitivity of detection for identifying tumor-infiltrating CD8 CAR T cells, ∼11,000 cells per mm3. These data suggest that the [18F]-TMP/eDHFR PET pair offers important advantages that could better allow investigators to monitor immune cell trafficking to tumors in patients.

Immunotherapy Using Chimeric Antigen Receptor-Engineered T Cells: A Novel Cellular Therapy with Important Implications for the Clinical Laboratory.

BACKGROUND: We have entered a new era of cancer therapy, with a number of immune-based therapies already used clinically as a standard of care. Adoptive cellular immunotherapy using T cells genetically modified with chimeric antigen receptors (CAR-T cells) represents a novel therapeutic approach. CAR-T cells have produced clinical responses in B-cell malignancies that are otherwise refractory to conventional therapies. Two CAR-T cell therapies obtained regulatory approval in 2017, with many more of these therapies under clinical development. CONTENT: This review focuses on the current state of adoptive cellular immunotherapy, specifically CAR-T cells, in the clinic and how this therapy differs from traditional small molecule and biologic therapies. Areas in which the clinical laboratory is affected by these novel therapies are discussed. Opportunities for the clinical laboratory to help guide these therapies are also highlighted. SUMMARY: The clinical laboratory will play an integral role in the care of patients undergoing adoptive cellular therapy with engineered T cells. There are many ways that this new therapeutic approach affects the clinical laboratory, and the clinical laboratory will likely play a critical role in managing patients that are treated with CAR-T cell therapy.

An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer.

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement in personalized cancer treatment. In this strategy, a patient's own T cells are genetically engineered to express a synthetic receptor that binds a tumor antigen. CAR T cells are then expanded for clinical use and infused back into the patient's body to attack and destroy chemotherapy-resistant cancer. Dramatic clinical responses and high rates of complete remission have been observed in the setting of CAR T-cell therapy of B-cell malignancies. This resulted in two recent FDA approvals of CAR T cells directed against the CD19 protein for treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Thus, CAR T cells are arguably one of the first successful examples of synthetic biology and personalized cellular cancer therapy to become commercially available. In this review, we introduce the concept of using CAR T cells to break immunological tolerance to tumors, highlight several challenges in the field, discuss the utility of biomarkers in the context of predicting clinical responses, and offer prospects for developing next-generation CAR T cell-based approaches that will improve outcome.

Results of ASERTAA, a Randomized Prospective Crossover Pharmacogenetic Study of Immediate-Release Versus Extended-Release Tacrolimus in African American Kidney Transplant Recipients.

BACKGROUND: Differences in tacrolimus dosing across ancestries is partly attributable to polymorphisms in CYP3A5 genes that encode tacrolimus-metabolizing cytochrome P450 3A5 enzymes. The CYP3A5*1 allele, preponderant in African Americans, is associated with rapid metabolism, subtherapeutic concentrations, and higher dose requirements for tacrolimus, all contributing to worse outcomes. Little is known about the relationship between CYP3A5 genotype and the tacrolimus pharmacokinetic area under the curve (AUC) profile in African Americans or whether pharmacogenetic differences exist between conventional twice-daily, rapidly absorbed, immediate-release tacrolimus (IR-Tac) and once-daily extended-release tacrolimus (LifeCycle Pharma Tac [LCPT]) with a delayed absorption profile. STUDY DESIGN: Randomized prospective crossover study. SETTING & PARTICIPANTS: 50 African American maintenance kidney recipients on stable IR-Tac dosing. INTERVENTION: Recipients were randomly assigned to continue IR-Tac on days 1 to 7 and then switch to LCPT on day 8 or receive LCPT on days 1 to 7 and then switch to IR-Tac on day 8. The LCPT dose was 85% of the IR-Tac total daily dose. OUTCOMES: Tacrolimus 24-hour AUC (AUC0-24), peak and trough concentrations (Cmax and Cmin), time to peak concentration, and bioavailability of LCPT versus IR-Tac, according to CYP3A5 genotype. MEASUREMENTS: CYP3A5 genotype, 24-hour tacrolimus pharmacokinetic profiles. RESULTS: ∼80% of participants carried the CYP3A5*1 allele (CYP3A5 expressers). There were no significant differences in AUC0-24 or Cmin between CYP3A5 expressers and nonexpressers during administration of either IR-Tac or LCPT. With IR-Tac, tacrolimus Cmax was 33% higher in CYP3A5 expressers compared with nonexpressers (P=0.04): With LCPT, this difference was 11% (P=0.4). LIMITATIONS: This was primarily a pharmacogenetic study rather than an efficacy study; the follow-up period was too short to capture clinical outcomes. CONCLUSIONS: Achieving therapeutic tacrolimus trough concentrations with IR-Tac in most African Americans results in significantly higher peak concentrations, potentially magnifying the risk for toxicity and adverse outcomes. This pharmacogenetic effect is attenuated by delayed tacrolimus absorption with LCPT. TRIAL REGISTRATION: Registered at ClinicalTrials.gov, with study number NCT01962922.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

MRI analysis of peripheral soft tissue composition, not body mass index, correlates with outcomes following anterior cruciate ligament reconstruction.

PURPOSE: To utilize MRI to assess the relationship between BMI, peripheral soft tissue composition about the knee, and surgical outcomes in patients undergoing primary ACL reconstruction. It was hypothesized that a fatty periarticular soft tissue composition may be better than BMI at predicting poor outcomes after ACL reconstruction. METHODS: Fifty-eight patients who underwent primary acute ACL reconstruction were identified from the surgical database and their data were retrospectively reviewed. Patients were selected based on availability of 2-year IKDC outcome scores, BMI data, and preoperative MRI studies. To approximate peripheral soft tissue composition, novel measurements of axial MRI images were employed using PACS ROI measurement tool. Relationships were assessed between IKDC outcome scores and measures of body habitus including BMI, total knee area, knee fatty-connective tissue area, and fatty-connective tissue to bone size ratio. RESULTS: The median BMI was 24.3 kg/m2 (range 18.5-36.9). Median IKDC score was 81.0 (range 46-100). BMI was correlated with total knee area (R = 0.72) and periarticular fat (R = 0.53). Neither continuous BMI (n.s.) nor total knee area (n.s.) was predictor of IKDC outcomes scores. Periarticular fatty-connective tissue trended towards predicting negative outcomes (n.s.). Periarticular fatty-connective tissue to bone size ratio was a significant negative predictor of IKDC scores (p = 0.03). Patients with more fat than bone on axial MRI (ratio > 1, N = 34) reported a lower mean IKDC score compared to patients with a ratio < 1 (N = 24) (77.2 vs. 87.7, p = 0.0028). The top quartile (N = 14) of these ratios reported a mean IKDC score of 68.9, compared to 87.3 of the bottom quartile (p = 0.0001). CONCLUSIONS: Periarticular soft tissue composition, as approximated by the novel MRI analysis of this study, is a better predictor of outcomes following ACL reconstruction than is BMI. This information can be utilized in guiding surgeon and patient expectations following surgery, either via a direct application of these measurements or heightened awareness of the importance of peripheral body habitus. LEVEL OF EVIDENCE: III.

Partial Wrist Denervation: The Evidence Behind a Small Fix for Big Problems.

Wrist denervation addresses symptomatic wrist pain without the morbidity and complication profile of more extensive surgical procedures aimed to correct the underlying pathology. The concept of wrist denervation is not new, but its practical application has been modified over the past 50 years. A variety of techniques have been described for various indications, with generally good results. In the United States, a simple, single incision partial denervation consisting of neurectomies of the anterior and posterior interosseous nerves is most commonly performed. Although data on this procedure are limited, most patients are satisfied with pain relief in the short term. There is no evidence that partial denervation procedures alter proprioception of the wrist, and this procedure shows promise as a good option for palliating pain without prolonged postoperative immobilization or leave from work. Preoperative injections do not seem to correlate well with postoperative results. Future studies are needed to assess the duration of relief and possible acceleration of underlying pathology.

Improving T Cell Expansion with a Soft Touch.

Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy but do not fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. A total of three different formulations of PDMS provided an extended proliferative phase in both CD4+-only and mixed CD4+-CD8+ T cell preparations. CD8+ T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-γ secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than that associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell based immunotherapy.

The Relationships Between Surface Measurements and Underlying Tendon Autograft Length for Upper Extremity Reconstructive Surgery.

PURPOSE: The availability of tendon grafts is an important consideration for successful upper extremity reconstructive surgery, including flexor or extensor tendon reconstructions, tendon transfers, and ligament reconstructions. Graft selection is based on availability, expendability, ease of harvest, and length. Given variations in patient height and extremity length, existing average values may provide suboptimal insight into actual tendon lengths available. The purpose of this study is, therefore, to pursue a method of estimating available donor tendon lengths based on easily measured anatomical surface landmarks. METHODS: Thirty cadaveric upper and lower extremity limbs were dissected and the length of commonly harvested tendon grafts including the palmaris longus, extensor indicis proprius, extensor digiti minimi, plantaris, and second long toe extensor was measured. Surface forearm length (from finger tip to cubital fossa) and surface fibular length (from lateral malleolus to fibular head) were also measured. Correlations between surface measurements and underlying tendon lengths were analyzed, and linear models were generated that predicted tendon length as a function of surface measurements. RESULTS: Surface measurements were correlated with underlying tendon length (R = 0.46 - 0.66). Linear models could predict tendon lengths based on surface measurements. A ratio of donor tendon length compared with the limb segment measured was established for each tendon and can be applied to estimate donor tendon length. For the upper extremity tendons, the multipliers for the palmaris longus, extensor indicis proprius, and extensor digiti minimi were 0.51, 0.20, and 0.18, respectively. Lower extremity tendon ratios for the plantaris and extensor digitorum longus were 0.69 and 0.60, respectively. CONCLUSIONS: Although length of available donor tendon can be a limiting variable at the time of surgery, surgeons may be better able to estimate underlying tendon lengths using easily obtained superficial measurements. CLINICAL RELEVANCE: Information obtained from these cadaveric measurements may aid in preoperative planning in hand and upper extremity surgery.

Rigid Patient Positioning is Unreliable in Total Hip Arthroplasty.

BACKGROUND: To our knowledge, no study has assessed the ability of rigid patient positioning devices to afford arthroplasty surgeons with ideal acetabular orientation throughout surgery. The purpose of this study is to use robotic arm-assisted computer navigation to assess the reliability of pelvic position in total hip arthroplasty performed on patients positioned with rigid positioning devices. METHODS: A prospective cohort of 100 hips (94 patients) underwent robotic-guided total hip arthroplasty in the lateral decubitus position from the posterior approach, 77 stabilized by universal lateral positioner, and 23 by peg board. Before reaming, computed tomography-templated computer software generated true values of pelvic anteversion and inclination based on the position of the robot arm registered to the patient's preoperative pelvic computed tomography. RESULTS: Mean alteration in anteversion and inclination values was 1.7° (absolute value, 5.3°; range, -20° to 20°) and 1.6° (absolute value, 2.6°; range, -8° to 10°), respectively. And 22% of anteversion values were altered by >10° and 41% by >5°. There was no difference between hip positioners used (P = .36). Anteversion variability was correlated with body mass index (P = .02). CONCLUSION: Despite the use of rigid patient positioning devices-a lateral hip positioner or peg board-this study reveals clinically important malposition of the pelvis in many cases, especially with regard to anteversion. These results show a clear need to pay particular attention to anatomic landmarks or computer-assisted techniques to assure accurate acetabular cup positioning. Patient positioning should not be solely trusted.

High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 µm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 µm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.