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 CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') 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-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta 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-zeta-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.

Regulation of DNA synthesis in Leydig cells obtained from neonatal pig testes.

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.

IL-10 modulates DSS-induced colitis through a macrophage-ROS-NO axis.

Breakdown of the epithelial barrier because of toxins or other insults leads to severe colitis. Interleukin-10 (IL-10) is a critical regulator of this, yet its cellular targets and mechanisms of action are not resolved. We address this here. Mice with a macrophage-selective deletion of IL-10Rα (IL-10Rα(Mdel)) developed markedly enhanced dextran sodium sulfate (DSS)-induced colitis that did not significantly differ from disease in IL-10(-/-) or IL-10Rα(-/-) mice; no impact of IL-10Rα deficiency in other lineages was observed. IL-10Rα(Mdel) colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Lamina propria macrophages (LPMφs) did not show numerical or phenotypic differences from controls, or a competitive advantage over wild-type cells. Proinflammatory cytokine production, and particularly tumor necrosis factor-α (TNF-α), was increased, although TNF-α neutralization failed to reveal a defining role for this cytokine in the aggravated disease. Rather, IL-10Rα(Mdel) LPMφs produced substantially greater levels of nitric oxide (NO) and reactive oxygen species (ROS) than controls. Inhibition of these had modest effects in wild-type mice, although they dramatically reduced colitis severity in IL-10Rα(Mdel) mice, and largely eliminated the differential effect of DSS in them. Therefore, the palliative actions of IL-10 in DSS-induced colitis predominantly results from its macrophage-specific effects. Downregulation of NO and ROS production are central to the protective actions of IL-10.

Induction of tolerance and immunity by redirected B cell-specific cytolytic T lymphocytes.

Chimeric receptors bearing ligand recognition domains linked to signaling regions from the T-cell receptor can redirect T lymphocytes against non-MHC-restricted targets. Cytolytic T lymphocytes (CTL) expressing these chimeric receptors are being tested in preclinical and clinical trials for activity in cancer, infectious diseases and autoimmunity. The chimeric receptors may incorporate antigenic epitopes previously unrecognized by the immune system. Whether a receptor-specific antibody response develops to these neoantigens and whether such a response inhibits therapeutic cell activity is unknown. We hypothesized that upon engagement of a chimeric receptor-specific B cell, receptor-modified CTL will be activated, lysing the B cell and inducing tolerance to the chimeric receptor rather than immunity. We demonstrate that receptor-modified CTL are indeed stimulated by cognate receptor-specific B cells, proliferate and produce cytokines in response and kill the B cells in vitro and in vivo. However, this is insufficient to induce full B-cell tolerance. Modified CTL induce a chimeric receptor-specific antibody response independent of any other source of antigen. Nevertheless, the CTL retain substantial activity even in the presence of saturating doses of receptor-specific antibody. Thus antichimeric receptor antibody responses need to be considered in the clinical use of chimeric receptor-modified T cells. However, the inhibitory activity of these antibodies may in cases be limited.

Antigen-specific targeting of CD8+ T cells with receptor-modified T lymphocytes.

Chimeric receptors that link ligand recognition domains, such as antibody Fv fragments, with TCR signaling domains can redirect T lymphocytes against MHC-unrestricted targets. Such receptor-modified T lymphocytes have shown promise in the treatment of infectious diseases and cancer. We hypothesized that receptor-modified T lymphocytes may also be designed to target antigen-specific T cells. We synthesized chimeric receptors consisting of the extracellular and transmembrane domains of the class I MHC H-2K(b) molecule linked to the signaling domains of either TCR-zeta, CD28 and zeta, or CD28, zeta, and lck. T lymphocytes modified to express these receptors and pulsed with antigenic peptide specifically killed precursor CTL. Cytolysis was efficient, even at effector:target ratios of less than one, and specific, selectively killing antigen-specific precursor CTL among a mixed population of T cells. Cytolysis required activation of the receptor-modified T cells, and did not occur with a signaling-deficient chimeric receptor. In contrast to precursor CTL, differentiated CTL proved resistant to lysis by the receptor-modified T cells. These data demonstrate the feasibility of redirecting T lymphocytes against antigen-specific T cells. Receptor-modified T cells expressing chimeric MHC receptors have potential application in autoimmune and alloimmune diseases.

The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes.

The TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the gamma-, delta-, epsilon-, and zeta-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR's ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR zeta-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-zeta receptor. Further, IAs-zeta stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the zeta-chain ITAMs, in the absence of the gamma, delta, and epsilon ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the zeta-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.

The effect of solvent/detergent-treated plasma on red cells stored in vitro.

BACKGROUND: The potential use of solvent/detergent-treated plasma (S/D plasma) in transfusion practice raises concerns about the cytolytic effects that any residual solvent and detergent in the virally inactivated blood component might have on units of red cells in vitro, if the two components are mixed during preparation. STUDY DESIGN AND METHODS: S/D plasma was mixed with variously processed units of stored red cells, in vitro, to evaluate the effect the residual solvent and detergent would have on cell membrane integrity. A paired protocol design was used in which half-units of red cells were exposed to S/D plasma (test), and the matched half-units were exposed to either the supernatant additive solution from the original red cell unit or standard fresh-frozen plasma (FFP) (control). After incubation for up to 5 days, the units were evaluated for evidence of hemolysis or changes in other red cell storage assays. RESULTS: This study showed that, for fresh additive solution red cells (AS-1), the 5-day storage plasma hemoglobin levels were comparable in the red cells exposed to S/D plasma (21 mg/dL) and in the paired half-units stored in the original AS-1 supernatant (31 mg/dL) (p > 0.05). Similar findings were recorded for stored AS-1 red cells (S/D plasma; 111 mg/dL vs. AS-1 supernatant, 147 mg/dL; p > 0.05); stored CPDA-1 red cells (S/D plasma, 133 mg/dL vs. FFP, 103 mg/dL; p > 0.05); frozen red cells (S/D plasma, 28 mg/dL vs. FFP, 18 mg/dL; p > 0.017); and stored irradiated AS-1 red cells (S/D plasma, 608 mg/dL vs. AS-1 supernatant, 726 mg/dL; p > 0.05). Comparable results were found for other assays, including levels of plasma potassium, osmotic fragility, and red cell antigen titer. CONCLUSION: These data show that S/D plasma does not induce red cell lysis even after 5 days of in vitro storage. These results are consistent with previous findings by this laboratory that platelets are not harmed by storage in S/D plasma. Red cells resuspended in S/D plasma and stored for up to 5 days maintain in vitro storage characteristics that are acceptable for the use of the cells in clinical transfusion practice.

Absence of D alloimmunization in D- pediatric oncology patients receiving D-incompatible single-donor platelets.

BACKGROUND: Guidelines are lacking for prophylaxis against D alloimmunization after D-incompatible platelet transfusion. A rational basis for the application of prophylaxis would be beneficial for institutions in which inventory constraints demand the administration of large numbers of D-incompatible platelets. STUDY DESIGN AND METHODS: A retrospective analysis was performed of all D-incompatible platelet transfusions administered at a pediatric research hospital over a 1.5-year period. Patients exclusively received single-donor WBC-reduced platelets and did not receive RhIg immunoprophylaxis. Numbers, source, ABO type, duration of serologic follow-up, and level of RBC contamination of D-incompatible transfusions were analyzed. All positive D serologies in the institution over a 3.5-year period were examined to determine cause and potential association with platelet transfusion. RESULTS: Thirty-five patients not receiving bone marrow transplant and seven bone marrow transplant patients received 490 and 255 D-incompatible transfusions, respectively, over 1.5 years. Patients had various diagnoses, predominantly malignancies. Seventy-nine percent of D-incompatible transfusions were ABO compatible. An estimated 2300 incompatible transfusions were performed over 3.5 years. No case of D alloimmunization was detected. CONCLUSIONS: D immunoprophylaxis is generally unnecessary in pediatric oncology patients receiving D-incompatible, WBC-reduced, single-donor platelets not visibly contaminated by RBCs. Further studies to validate these observations in the pediatric population and to extend them to other population groups are warranted.

Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors in T lymphocytes.

Adoptive immunotherapy using receptor-modified T lymphocytes has shown promise in preclinical studies for the treatment of infectious and malignant diseases. These modified T cells express chimeric receptors that link ligand recognition and signal transduction domains in a single gene product. Typically, a single chain Fv fragment is genetically attached to the cytoplasmic domain of the T-cell receptor (TCR) zeta chain. Modulating the signaling characteristics of chimeric receptors will be important for their application to human immunotherapy. It was hypothesized that linking coreceptor and costimulatory signaling motifs together with the zeta signaling domain will enhance receptor function. The present study compares signaling characteristics of 9 single-chain receptors consisting of the H-2K(b) extracellular and transmembrane domains and various combinations of T cell signal transduction domains. Signal transduction regions studied include the TCR zeta chain, the CD4 coreceptor, the lck protein tyrosine kinase, and the CD28 costimulatory receptor. Biochemical characteristics of the receptors, analyzed using calcium flux, receptor, and ZAP-70 phosphorylation, and lck association may be predicted from the known functions of receptor constituents. The combination of zeta together with coreceptor and costimulatory function in a single receptor maximizes chimeric receptor sensitivity and potency. Combining zeta with either the costimulatory or coreceptor function independently also enhances receptor function, though to a lesser extent. It is therefore possible to link TCR, coreceptor, and costimulatory activities in a single functional entity using modular domains. Such receptors demonstrate distinct signaling properties and should prove useful in the development of chimeric receptors for therapeutic purposes.

Removal of anaphylatoxins C3a and C5a and chemokines interleukin 8 and RANTES by polyester white cell-reduction and plasma filters.

BACKGROUND: A few bedside polyester white cell (WBC)-reduction filters have been shown to scavenge C3a anaphylatoxin from stored blood components. One has been shown to remove the chemokines interleukin (IL)-8 and RANTES, but not the proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor alpha. Removal by any filter of the anaphylatoxin C5a or the soluble membrane attack complex (SC5b-9) has not been studied. Further, the ability of other filters to scavenge these biologic response modifiers (BRM) is not known. Four WBC-reduction filters and one plasma filter were studied for their ability to remove IL-8, RANTES, IL-1 beta, C3a, C5a, and SC5b-9. STUDY DESIGN AND METHODS: Plasma was obtained either as freshly thawed fresh-frozen plasma, fresh-frozen plasma thawed and stored for 5 days, or platelet-poor supernatant. Cell-poor plasma was obtained and samples were taken before and after filtration through the various filters Levels of IL-1 beta, IL-8, RANTES, C3a, and SC5b9 were quantitated by enzyme immunoassay. To evaluate filter scavenging of C5a, an in vitro model was developed to generate high levels of C5a in plasma by activating plasma with zymosan. RESULTS: Levels of C3a, C5a, IL-8, and RANTES were reduced by filtration through two bedside platelet WBC-reduction filters, a plasma filter, and a prestorage red cell WBC-reduction filter, but not following filtration through a prestorage platelet WBC-reduction filter. For some BRMs and filters, however, evidence of filter saturation was seen. IL-1 beta was not removed by any of the filters tested. CONCLUSION: Some, but not all, bedside polyester filters and prestorage polyester filters can remove IL-8, RANTES, C3a, and C5a from units of plasma or platelets. Improved biomaterial engineering of these and other filters could maximize scavenging of BRMs and potentially diminish the adverse reactions associated with their infusion during transfusion.

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.