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Abstract: In September 2005, an National Institutes of Health-sponsored workshop was held in Atlanta, Georgia, which focused on non-human primate transplantation tolerance models. The aim of this workshop was an open exchange of techniques and assays applicable to this important translational transplantation model. A major focus of the workshop was the development of assays that are capable of monitoring immune status after transplant. These include mixed lymphocyte reaction assays (using both tritiated thymidine and carboxyfluorescein succinimidyl ester to assess T-cell proliferation), large-scale production of dendritic cells for immune analysis, the molecular analysis of graft rejection and acceptance using single-plex RNA analysis, and multiplex flow cytometric and gene array analysis, as well as sophisticated multi-color flow cytometric profiling of immunocyte subpopulations and cytokine expression patterns. This review will summarize the progress presented at the workshop toward the application of these techniques to immune analysis in non-human primates and discuss their relative contributions to ongoing evaluation of immune tolerance after transplantation. [Copyright &y& Elsevier]

: Objectives1) To study the mechanisms of anemia, erythroid hyperplasia, and red blood cell (RBC) clearance in murine models of sickle cell disease (Sickle) and β-thalassemia (Th1/Th1); 2) To determine the contribution of the phospholipid scramblase enzyme to phosphatidylserine (PS) exposure and RBC death in Sickle and Th1/Th1 mice.: MethodsWe used a combination of flow-cytometric analysis and assays for phospholipid remodeling to determine the extent and sites of erythroid hyperplasia, PS exposure, and cell death.: Results1) Sickle RBCs have a much shorter half-life than Th1/Th1 RBCs (0.8 days vs 11 days). A significant proportion of Th1/Th1 peripheral reticulocytes mature into erythrocytes, however, ∼fivefold fewer Sickle reticulocytes mature. While erythroid hyperplasia exists in both Sickle and Th1/Th1 mice, Th1/Th1 produce fourfold more RBCs than necessary to maintain steady state, while Sickle produce no excess RBCs. 2) 61% of Sickle and 34% of Th1/Th1 RBCs are scramblase+ as measured by internalization assays of the fluorescent phospholipid NBD-PC. The majority of NBD-PC+ RBCs are also annexin-V+, supporting a mechanistic link between scramblase activity and PS exposure. A proportion of both reticulocytes and older RBCs in Sickle and Th1/Th1 mice have active scramblase, and the degree of scramblase activation in these strains correlates with the propensity for RBC death.: ConclusionsSickle and Th1/Th1 mice are both anemic, with significant erythroid hyperplasia. Th1/Th1 mice display ineffective erythropoiesis while Sickle mice show rapid peripheral destruction of RBCs. PS exposure and phospholipid scramblase activity serve as markers of RBCs with altered phospholipid asymmetry and greater propensity for cell death. [Copyright &y& Elsevier]

Therapeutic T cell engineering has recently garnered widespread interest because of the success of CD19 chimeric antigen receptor (CAR) therapy. CARs are synthetic receptors for antigen that redirect the specificity and reprogram the function of the T cells in which they are genetically introduced. CARs targeting CD19, a cell surface molecule found in most leukemias and lymphomas, have yielded high remission rates in patients with chemorefractory, relapsed disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. The toxicities of this treatment include B cell aplasia, cytokine release syndrome (CRS), and neurotoxicity. Although reversible in most instances, these toxicities may require specific medical interventions, including transfer to intensive care to treat severe CRS. Guidelines for managing these toxicities are emerging. The recent report of a nonhuman primate model for CRS is poised to help advance the management of this syndrome. Finally, new engineering modalities, based on the use of targeted nucleases like CRISPR, may further enhance the efficacy and safety of CAR T cells. [ABSTRACT FROM AUTHOR]

Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3 expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile—desirable properties that support the possibility of off-the-shelf Treg therapeutics. [ABSTRACT FROM AUTHOR]

Therapeutic T cell engineering has recently garnered widespread interest because of the success of CD19 chimeric antigen receptor (CAR) therapy. CARs are synthetic receptors for antigen that redirect the specificity and reprogram the function of the T cells in which they are genetically introduced. CARs targeting CD19, a cell surface molecule found in most leukemias and lymphomas, have yielded high remission rates in patients with chemorefractory, relapsed disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. The toxicities of this treatment include B cell aplasia, cytokine release syndrome (CRS), and neurotoxicity. Although reversible in most instances, these toxicities may require specific medical interventions, including transfer to intensive care to treat severe CRS. Guidelines for managing these toxicities are emerging. The recent report of a nonhuman primate model for CRS is poised to help advance the management of this syndrome. Finally, new engineering modalities, based on the use of targeted nucleases like CRISPR, may further enhance the efficacy and safety of CAR T cells. [ABSTRACT FROM AUTHOR]