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Tisagenlecleucel is a chimeric antigen receptor–T cell therapy that facilitates the killing of CD19+ B cells. A model was developed for the kinetics of tisagenlecleucel and the impact of therapies for treating cytokine release syndrome (tocilizumab and corticosteroids) on expansion. Data from two phase II studies in pediatric and young adult relapsed/refractory B cell acute lymphoblastic leukemia were pooled to evaluate this model and evaluate extrinsic and intrinsic factors that may impact the extent of tisagenlecleucel expansion. The doubling time, initial decline half‐life, and terminal half‐life for tisagenlecleucel were 0.78, 4.3, and 220 days, respectively. No impact of tocilizumab or corticosteroids on the expansion rate was observed. This work represents the first mixed‐effect model‐based analysis of chimeric antigen receptor–T cell therapy and may be clinically impactful as future studies examine prophylactic interventions in patients at risk of higher grade cytokine release syndrome and the effects of these interventions on chimeric antigen receptor–T cell expansion.

Limited cell numbers in umbilical cord blood (UCB) grafts present a major impediment to favorable outcomes in adult transplantation, largely related to delayed or failed engraftment. The advent of UCB transplantation (UCBT) using two grafts successfully circumvents this obstacle, despite the engraftment of only one unit. Preclinical models suggested that the addition of UCB T cells at the time of transplant can enhance engraftment. We tested whether ex vivo activation by CD3/CD28 costimulation and expansion of T cells from a single UCB graft would be safe and feasible in adults with advanced hematologic malignancies, with an overall objective of optimizing engraftment in single unit UCBT. In this phase 1 study, recipients of single UCB units were eligible if the unit was stored in two adequate fractions. Dose limiting toxicity was defined as grade 3 or grade 4 GVHD within 90 days of UCBT. Four patients underwent UCBT; all were treated at the first dose level (10(5) cells/kg). At the 10(5) cells/kg dose level two subjects experienced grade 3 intestinal GVHD, thus meeting stopping criteria. For three subjects, neutrophil engraftment was early (12, 17, and 20 days), while one subject experienced primary graft failure. We observed early donor T cell trafficking and found that expanded T cells produced supraphysiologic levels of cytokines relevant to engraftment and to lymphoid differentiation and function. Taken together, these preliminary data suggest rapid engraftment in recipients of a single UCBT combined with relatively low doses of activated T cells, though potentially complicated by severe GVHD.

Background Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. Methods Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 108 cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. Results Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 1012 VRX496 proviral copies were administered to these 65 subjects. Conclusions Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors. Copyright © 2013 John Wiley & Sons, Ltd.

Cultured rat cerebellar granule cells are resistant to the excitotoxic effects of N-methyl-d-aspartate (NMDA) and non-NMDA receptor agonists under three conditions: 1) prior to day seven in vitro when cultured in depolarizing concentrations of potassium [25 mM]; 2) at any time in vitro when cultured in non-depolarizing concentrations of potassium [5 mM]; and 3) when neurons, cultured in depolarizing concentrations of potassium [25 mM] for eight days in vitro, are pretreated with a subtoxic concentration of NMDA. The focus of this paper is to determine: a) whether the resistance to excitotoxicity by NMDA and non-NMDA receptor agonists is due to a decreased intracellular calcium [Ca++]i response to glutamate receptor agonists in cultured rat cerebellar granule cells; or b) whether [Ca++]i levels induced by the agonists are similar to those observed under excitotoxic conditions. Granule cells, matured in non-depolarizing growth medium, treated with glutamate resulted in an increase in [Ca++]i followed by a plateau that remained above baseline in virtually all neurons that responded to glutamate. The response was rapid in onset (

Telomerase activity and the regulation of telomere length are factors which have been implicated in the control of cellular replication. These variables have been examined during human lymphocyte development, differentiation, activation, and aging. It was found that telomere length of peripheral blood CD4+ T cells decreases with age as well as with differentiation from naive to memory cells in vivo, and decreases with cell division in vitro. These results provide evidence that telomere length correlates with lymphocyte replicative history and residual replicative potential. In contrast, telomere length appears to increase during tonsil B-cell differentiation and germinal center (GC) formation in vivo. It was also found that telomerase activity is highly regulated during T-cell development and B-cell differentiation in vivo, with high levels of telomerase activity expressed in thymocytes and GC B cells, and low levels of telomerase activity in resting mature peripheral blood lymphocytes. Finally, resting lymphocytes retain the ability to upregulate telomerase activity upon activation, and this capacity does not appear to decline with age. Although the precise role of telomerase in lymphocyte function remains to be elucidated, telomerase may contribute to protection from telomere shortening in T and B lymphocytes, and may thus play a critical role in lymphocyte development, differentiation and activation. The future study of telomerase and its regulation of telomere length may enhance our understanding of how the replicative lifespan is regulated in lymphocytes.

Peripheral blood mononuclear cells from many asymptomatic HIV-infected patients exhibit defects in cytokine production and impaired proliferative responses in vitro but the mechanisms underlying this subclinical immune deficiency are controversial. To determine whether abnormalities in the earliest events following receptor engagement may help to explain the in vitro immune dysfunction, we measured the inducibility of the early activation marker CD69 in T cells from asymptomatic HIV-infected individuals in response to stimulation with anti-CD2 or anti-CD3 mAb. In a whole blood assay, we found that induction of CD69 was markedly impaired in CD4+ T cells from later-stage HIV-infected patients (CD4 counts 200-400/mm3) compared to uninfected controls. Among early stage patients (CD4 > 400/mm3), a subset (29%) had impaired CD69 induction. CD69 responses were equally depressed after stimulation through the CD3 or CD2 receptor pathways. Survey of a panel of immunophenotypic markers and propensity for apoptosis demonstrated a significant association between depressed induction of CD69 and decreased percentages of CD4+CD26+ and CD4+CD95+ cells but no association with the level of apoptosis. These data indicate that defects in T lymphocyte activation through CD3 and CD2 can be measured within hours of receptor stimulation in asymptomatic HIV-infected individuals and might be useful to monitor as an indicator of immune function in these patients.

Engagement of CD28 on T cells provides a co-stimulatory signal necessary for T cell activation and differentiation. Recent findings suggest that priming of T helper (Th)2 cells is more dependent on CD28 activation that Th1 cells. The present study examines whether mice that lack expression of CD28 as a result of gene targeting are capable of generating a Th2 response characteristic during infection with the intravascular trematode parasite Schistosoma mansoni. Mutant and control mice were either inoculated in the footpad with S. mansoni eggs (a potent inducer of a Th2 response) or infected percutaneously with the parasite. Draining lymph nodes (after footpad injection) or spleen cells (after natural infection) were harvested at 12 days and 8 weeks, respectively, and examined for cytokine responses to egg antigens. CD28-deficient mice (-/-) generated diminished egg antigen-driven interleukin (IL)-4 and IL-5 production (by 5- to 17-fold, respectively) compared to CD28-expressing (+/+) littermates. In contrast, lymphocyte proliferation and interferon (IFN)-gamma production to egg antigens were equivalent for mutant and control mice. Infected CD28-/- mice also had reduced immunoglobulin secretion. Serum levels of parasite antigen-specific IgG1 and polyclonal IgE were significantly diminished in CD28-/- compared to CD28+/+ mice. Lack of CD28 expression had no effect on granuloma formation around eggs trapped in the liver, but increased susceptibility of mice to primary schistosomiasis infection. These studies indicate that CD28 activation contributes to T cell priming required for generation of a Th2 response to an intravascular dwelling helminth parasite.

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4+ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4+ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-gamma (IFN-gamma) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-gamma production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4+ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.

The use of flow cytometry to identify and isolate cells by surface antigen expression was a fundamental advance for immunologists and hematologists and now has an increasing role in the clinical practice of medicine. Similarly, the use of the flow cytometer to study lymphocyte activation and cellular metabolism has proved to be a powerful technique. Knowledge of signal transduction mechanisms has progressed to a point where clinical applications of signal transduction measurements can now be anticipated. The development of a number of fluorescent probes for the study of intracellular ion concentrations in single, viable cells has permitted a substantial advance in the understanding of many areas of immunology. It is well established that changes in intracellular free calcium concentration play a key role in some, but not all forms of B and T cell activation

Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation.

The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). This unit describes flow cytometric protocols using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer.

The binding of antibody to the CD4 molecule inhibits mobilization of cytoplasmic free calcium ([Ca2+]i) in response to CD3 cross-linking on resting T cells. Similarly, when CD3 and CD4 are independently and simultaneously cross-linked, calcium mobilization is inhibited when compared to that induced by cross-linking CD3 alone. In contrast, when anti-CD4 and anti-CD3 are cross-linked together, calcium mobilization is substantially higher than from CD3 cross-linking alone. A heteroconjugate consisting of covalently bound CD3 and CD4 monoclonal antibodies (mAb) retains the ability to mobilize [Ca2+]i in CD4 cells at protein concentrations approximately two orders of magnitude lower than the free CD3 mAb, and the activity of the heteroconjugate is inhibitable by free CD4 mAb. The CD3/CD4 heteroconjugate also shows significantly greater activity in stimulation of inositol phosphate IP1, IP2 and IP3 synthesis in T cells than the CD3 mAb alone, and again the activity is inhibited by free CD4 mAb. The activity of the CD3/CD4 heteroconjugate is not simply due to oligomerization, since CD3/CD3 or CD4/CD4 homoconjugates or homoconjugate mixtures did not show increased activity. Other heteroconjugates (CD3/CD5 and CD3/CD28) were not different than the CD3/CD3 homoconjugate in their ability to increase [Ca2+]i. Purified CD4 T cells that do not respond to CD3 mAb in solution do respond to the CD3/CD4 heteroconjugate in solution by proliferating in the presence of a CD28 mAb, with a significant fraction of CD4 cells entering the second cycle within the first three days of stimulation. The CD3/CD4 heteroconjugate co-modulates the CD3 and CD4 receptors, indicating that the heteroconjugate is not simply anchoring the T cell receptor to the T cell surface like anti-CD3 on a solid surface. These results suggest that CD4 plays an active role in signal transduction when brought into close physical proximity to the CD3/T cell receptor complex during major histocompatibility complex class II-restricted antigen presentation.

The relationship between the increase of intracellular free Ca2+ concentration ([Ca2+]i) in resting T cells after stimulation with monoclonal antibody (mAb) to CD2 (E rosette receptor) and the subsequent proliferation response was investigated. Although the combination of 9.6 plus 9-1 mAb to CD2 was both mitogenic and induced an increase in [Ca2+]i, cross-linking of an individual CD2 mAb on the cell surface induced an increase in [Ca2+]i without directly stimulating T cell proliferation. The [Ca2+]i response from cross-linking an individual mAb was not epitope dependent, since 21 of 21 mAb to CD2 were effective when cross-linked with a polyclonal goat anti-mouse Ig second step. The kinetics of calcium mobilization was highly dependent upon the procedure for cross-linking, since the binding of biotin-conjugated 9.6 mAb followed by avidin gave a large and rapid response, whereas cross-linking of 9.6 with an anti-kappa mAb, 187.1, caused a minimal response, and the cross-linking of 9.6 followed by a polyclonal goat anti-mouse Ig gave an intermediate response. In addition, ligation of CD2 by rosetting with sheep red blood cells alone was sufficient to cause increased [Ca2+]i. In functional studies only the procedures associated with minimal CD2 cross-linking induced proliferation of resting T cells in combination with interleukin (IL)2. The proliferation also required IL 1 or accessory cells. Cross-linking 9.6 on the cell surface also enhanced proliferation in the presence of phorbol myristate acetate or a CD28 mAb, 9.3, under conditions that were accessory cell independent. In contrast to the proliferation following stimulation with 9.6 plus 9-1, the combination of 9.6 plus 9-1 F(ab')2 fragments lost mitogenic activity. The 9.6 plus 9-1 F(ab')2 combination was similar to 9.6 cross-linking in that either could induce responsiveness to recombinant IL2 or CD28 mAb 9.3. The combination of 9.6 plus 9-1 F(ab')2 fragments was still able to increase [Ca2+]i in T cells with slow kinetics. Together these results suggest that the binding of mAb to CD2 under conditions that cause a slow rather than a rapid increase in [Ca2+]i is associated with T cell activation. Furthermore, they suggest that in studies of T cell activation, sheep erythrocyte rosette formation should not be used to isolate T cells since rosetting may effect [Ca2+]i.