Terrence L. Geiger

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia

To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8α, and the signaling domain of CD3ζ. We compared the antileukemic activity mediated by a novel receptor (‘anti-CD19-BB-ζ’) containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-ζ receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-ζ lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-ζ-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.

A Kinetic and Dynamic Analysis of Foxp3 Induced in T Cells by TGF-β

TGF-β induces Foxp3 expression in stimulated T cells. These Foxp3+ cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3+ regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-β up-regulated Foxp3 in CD4+Foxp3− T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1–2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-β led to a loss of Foxp3 expression after an ∼4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3− cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3− cells were detected by 28 days after transfer. However, some Foxp3+ cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3+ iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3− cells, Foxp3+ iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naïve-stimulated T cells is transient in vitro, dependent on TGF-β activity within a highly restricted window after activation and continuous TGF-β presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.

Mitigation of Experimental Allergic Encephalomyelitis by TGF-β Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance

Stimulation of naive T lymphocytes in the presence of IL-2 and TGF-β induces the regulatory transcription factor Foxp3, which endows the cells with regulatory functions. To better understand the properties and therapeutic potential of these induced regulatory T cells (iTreg), we examined their immunomodulatory properties in myelin oligodendroglial glycoprotein-induced experimental allergic encephalomyelitis (MOG-EAE). Adoptively transferred iTreg were as potent as natural Foxp3+ Treg in preventing EAE development, and were active both prophylactically and after priming. The iTreg migrated into the CNS in quantity, skewing the ratio of regulatory to effector T lymphocytes. IL-10−/− iTreg failed to suppress disease, demonstrating a critical role for iTreg IL-10 production in their therapeutic activity. MOG-specific T cells from iTreg treated animals were anergic. The cells failed to proliferate in response to Ag except in the presence of exogenous IL-2, and did not secrete or secreted reduced amounts of IL-2, IFN-γ, and IL-17. MOG-specific T cells were not wholly unresponsive though, as they did secrete IL-10 after stimulation. To determine whether iTreg-mediated tolerance was infectious, fostering the development of T lymphocytes that could independently suppress EAE, we purged draining lymph node cells from MOG-immunized, iTreg treated mice of the administered iTreg, and transferred the remaining cells to Ag-inexperienced mice. The transferred cells were able to block EAE development. Thus iTreg are highly potent suppressors of autoimmune encephalomyelitis, and act in an IL-10 dependent manner both through the induction of anergy in effector T cells and through the infectious induction of protective T lymphocytes able to independently suppress disease development.

Regulation of ZAP-70 Activation and TCR Signaling by Two Related Proteins, Sts-1 and Sts-2

T cells play a central role in the recognition and elimination of foreign pathogens. Signals through the T cell receptor (TCR) control the extent and duration of the T cell response. To ensure that T cells are not inappropriately activated, signaling pathways downstream of the TCR are subject to multiple levels of positive and negative regulation. Herein, we describe two related proteins, Sts-1 and Sts-2, that negatively regulate TCR signaling. T cells from mice lacking Sts-1 and Sts-2 are hyperresponsive to TCR stimulation. The phenotype is accompanied by increased Zap-70 phosphorylation and activation, including its ubiquitinylated forms. Additionally, hyperactivation of signaling proteins downstream of the TCR, a marked increase in cytokine production by Sts1/2(-/-) T cells, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis is observed. Therefore, Sts-1 and Sts-2 are critical regulators of the signaling pathways that regulate T cell activation.

A one-step large-scale method for T- and B-cell depletion of mobilized PBSC for allogeneic transplantation

BACKGROUND The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. METHODS T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. RESULTS The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19x10(10) (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log(10) depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log(10) depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34(+) stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2x10(8) (range 2-10x10(8)). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. DISCUSSION Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft.

Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire

BackgroundRecent advances in massively parallel sequencing have increased the depth at which T cell receptor (TCR) repertoires can be probed by >3log10, allowing for saturation sequencing of immune repertoires. The resolution of this sequencing is dependent on its accuracy, and direct assessments of the errors formed during high throughput repertoire analyses are limited.ResultsWe analyzed 3 monoclonal TCR from TCR transgenic, Rag-/- mice using Illumina® sequencing. A total of 27 sequencing reactions were performed for each TCR using a trifurcating design in which samples were divided into 3 at significant processing junctures. More than 20 million complementarity determining region (CDR) 3 sequences were analyzed. Filtering for lower quality sequences diminished but did not eliminate sequence errors, which occurred within 1-6% of sequences. Erroneous sequences were pre-dominantly of correct length and contained single nucleotide substitutions. Rates of specific substitutions varied dramatically in a position-dependent manner. Four substitutions, all purine-pyrimidine transversions, predominated. Solid phase amplification and sequencing rather than liquid sample amplification and preparation appeared to be the primary sources of error. Analysis of polyclonal repertoires demonstrated the impact of error accumulation on data parameters.ConclusionsCaution is needed in interpreting repertoire data due to potential contamination with mis-sequence reads. However, a high association of errors with phred score, high relatedness of erroneous sequences with the parental sequence, dominance of specific nt substitutions, and skewed ratio of forward to reverse reads among erroneous sequences indicate approaches to filter erroneous sequences from repertoire data sets.

Early complications in children with acute lymphoblastic leukemia presenting with hyperleukocytosis

The optimal management of childhood acute lymphoblastic leukemia (ALL) with hyperleukocytosis is unclear, largely because the risk of leukostasis‐related complications is poorly characterized.

Purification of human natural killer cells using a clinical-scale immunomagnetic method

BACKGROUND Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

A large-scale method for the selective depletion of αβ T lymphocytes from PBSC for allogeneic transplantation

BackgroundWe sought to develop a method for the clinical large-scale depletion of αβ T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and γδ T lymphocytes.MethodsTherefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-αβ T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS® device.ResultsThe median T-cell depletion was 3.9 log (range 3.5–4.1 log). The recovery of the γδ and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%.DiscussionThis method had no negative influence on the in vitro colony formation of stem cells, and transplantation of αβ-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r null) mice resulted in a rapid eng...

Premedication with acetaminophen or diphenhydramine for transfusion with leucoreduced blood products in children

Febrile non‐haemolytic or allergic reactions occur in 0·1–30% of transfusions; physicians often premedicate patients with acetaminophen or diphenhydramine to prevent these reactions. The effectiveness of this practice has not been demonstrated. In this retrospective review of all transfusions at our institution during 2002, 385 patients received 7900 evaluable leucoreduced, irradiated blood products (4280 single‐donor apheresis platelets and 3620 packed red blood cells). Febrile reactions occurred in 0·95% of 4108 transfusions with, and 0·53% of 3792 transfusions without, acetaminophen premedication. Allergic reactions occurred in 0·90% of 4315 transfusions with, and 0·56% of 3585 transfusions without, diphenhydramine premedication. In a multivariate analysis that adjusted for age, patient category, transfusion location, product, transfusion history, and reaction history, premedication with acetaminophen was associated with a statistically non‐significant increase in the odds of a febrile reaction (odds ratio 1·74; 95% confidence interval 0·71–4·23; P = 0·22), and diphenhydramine with a non‐significant increase in allergic reactions (odds ratio 1·74; 95% confidence interval 0·99–3·06; P = 0·054). Reactions occurred in only 1·3% of the 518 transfusions to patients with a history of two or more prior reactions. Febrile and allergic transfusion reactions were rare in paediatric patients transfused with leucoreduced, irradiated blood products, whether premedication was used or not.