Activation of Acute Lymphocytic Leukemia Blasts with CpG ODN, IL-4 and CD40 Ligand Facilitates Enhanced Anti-Leukemic CTL Responses

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3907-3907
Author(s):  
Dorit Fabricius ◽  
Lucia Breckerbohm ◽  
Manon Queudeville ◽  
Angelika Vollmer ◽  
Sarah M. Eckhoff ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Combined Treatment ◽  
Cd40 Ligand ◽  
Term Survival ◽  
Hodgkin's Lymphomas ◽  
Novel Therapeutic

Abstract Abstract 3907 Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phenotypic and functional characterization of T-BAM (CD40 ligand)+ T- cell non-Hodgkin's lymphoma

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 866-872 ◽  
Author(s):  
G Inghirami ◽  
S Lederman ◽  
MJ Yellin ◽  
A Chadburn ◽  
L Chess ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Functional Characterization ◽  
Cd40 Ligand ◽  
B Cell Differentiation ◽  
Helper Function ◽  
Unique Distribution ◽  
Hodgkin's Lymphomas

Abstract The precise mechanisms regulating T-helper function have been intensively investigated. We and others have recently identified a new T-cell-B-cell-activating molecule called T-BAM that directs B-cell differentiation by interacting with the CD40 molecule on B cells. Using a specific monoclonal antibody against T-BAM (5C8), we have previously shown that T-BAM expressing T cells are predominantly CD4+CD8- and in normal lymphoid tissue have a unique distribution. However, no information has been obtained regarding the phenotype and functional properties of human neoplastic T cells. Therefore, we investigated T- BAM expression immunohistochemically in 87 well-characterized T-cell non-Hodgkin's lymphomas and lymphoid leukemias (LL). We found that 21/81 neoplasms expressed detectable T-BAM and these positive tumors belong almost exclusively to the CD4+CD8- subtype. In addition, to determine whether T-BAM expression could be induced on T-BAM-LL cells, we activated T-BAM-LLs in vitro and showed that T-BAM could be upregulated only in CD4+CD8- tumors. Our studies clearly show that T- BAM is constitutively expressed in a large number of T-cell neoplasms with a relative mature phenotype (CD4+CD8-) and that only CD4+ neoplastic T cells can be induced in vitro to express this molecule. Additional studies are necessary to identify the biologic significance of T-BAM expression and its potential and clinical implications.

scholarly journals Phenotypic and functional characterization of T-BAM (CD40 ligand)+ T- cell non-Hodgkin's lymphoma

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 866-872 ◽  
Author(s):  
G Inghirami ◽  
S Lederman ◽  
MJ Yellin ◽  
A Chadburn ◽  
L Chess ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Functional Characterization ◽  
Cd40 Ligand ◽  
B Cell Differentiation ◽  
Helper Function ◽  
Unique Distribution ◽  
Hodgkin's Lymphomas

The precise mechanisms regulating T-helper function have been intensively investigated. We and others have recently identified a new T-cell-B-cell-activating molecule called T-BAM that directs B-cell differentiation by interacting with the CD40 molecule on B cells. Using a specific monoclonal antibody against T-BAM (5C8), we have previously shown that T-BAM expressing T cells are predominantly CD4+CD8- and in normal lymphoid tissue have a unique distribution. However, no information has been obtained regarding the phenotype and functional properties of human neoplastic T cells. Therefore, we investigated T- BAM expression immunohistochemically in 87 well-characterized T-cell non-Hodgkin's lymphomas and lymphoid leukemias (LL). We found that 21/81 neoplasms expressed detectable T-BAM and these positive tumors belong almost exclusively to the CD4+CD8- subtype. In addition, to determine whether T-BAM expression could be induced on T-BAM-LL cells, we activated T-BAM-LLs in vitro and showed that T-BAM could be upregulated only in CD4+CD8- tumors. Our studies clearly show that T- BAM is constitutively expressed in a large number of T-cell neoplasms with a relative mature phenotype (CD4+CD8-) and that only CD4+ neoplastic T cells can be induced in vitro to express this molecule. Additional studies are necessary to identify the biologic significance of T-BAM expression and its potential and clinical implications.

scholarly journals Corticosteroids Do Not Influence the Efficacy and Kinetics of CAR-T Cells for B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 228-228 ◽  
Author(s):  
Shuangyou Liu ◽  
Biping Deng ◽  
Jing PAN ◽  
Zhichao Yin ◽  
Yuehui Lin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
High Dose ◽  
Car T Cells ◽  
Steroid Group ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Kinetics Of

Cytokine release syndrome (CRS) is the most prominent and potentially life-threatening toxicity caused by chimeric antigen receptor (CAR) T cell therapy, therefore, effectively controlling severe CRS is critical to ensure patient safety. Tocilizumab, an interleukin-6 receptor antagonist, has been widely used to treat CRS, whereas it is not clear if corticosteroids could be as another optimal choice for managing CRS. We applied corticosteroids instead of tocilizumab as the first-line agent to control CRS in patients with relapsed/refractory B-cell acute lymphoblastic leukemia during CAR-T therapy. The impacts of steroids on treatment efficiency and kinetics of CAR-T cells were assessed by comparing two groups of patients who did (42 cases) or did not (26 cases) receive steroids. Patients followed up less than one month (went to other hospitals for transplantation or died within one month) were excluded. Treatment effects were evaluated on day 30 after T-cell infusion and then monthly in follow-up patients. Minimal residual disease (MRD) was detected by multiparameter flow cytometry (FCM) and quantitative PCR for fusion genes. The dynamic monitoring of CAR-T cells was performed through flow cytometric quantitation of FITC+CD3+ T cells. B-cell aplasia (BCA) was assayed by FCM. Dexamethasone or methylprednisolone or both (alternately) were administrated. Dexamethasone was used in most cases especially for patients with neurologic symptoms; methylprednisolone was preferred for patients with pulmonary or liver dysfunction, and patients accepting high dose steroids. Steroids started with low dose and could be increased if symptoms were not resolved, for severe CRS, steroids would be escalated up to dexamethasone 20mg/m2/d or more higher up to methylprednisolone 10mg/kg/d. Once CRS was improved, steroids were rapidly reduced and stopped. A total of 68 patients (28 adults and 40 children younger than 18 years) were included, 22 (32.4%) presented with extramedullary diseases (EMD), bone marrow blasts in patients without EMD varied between 5%-96.5%, 31 (45.6%) patients had an allogeneic transplantation, 54 (79.4%) cases received CD19-specific and 14 (20.6%) received CD22-specific CAR-T therapy. Forty-two (61.8%) cases, including all (10) of grade III CRS, 68.2% (30/44) of grade II CRS and 2 patients with no CRS but with GVHD (1 case) or neurotoxicity (1 case), were administered steroids, among them, 23/42 (54.8%) received high dose steroids (>10mg/m2/d dexamethasone or equivalent), the duration of steroid use was 1-16 days (78.6% <= 7 days); whereas 26 (38.2%) patients were not given any steroids but the supportive care. We found that there was no difference either in complete remission (CR) rate (95.2% vs 92.3%, p=.344) or in MRD negative CR rate (80.0% vs 79.2%, p=.249) between steroid and non-steroid group, verified that corticosteroids even high dose steroids did not influence the treatment response. Furthermore, we investigated the dynamics of CAR-T cells. Firstly, the expansion of CAR-T cells in peripheral blood (PB) was evaluated, the average CAR-T cell counts in steroid group were significantly higher than those in non-steroid group on D11 (p=.0302), D15 (p=.0053), D20 (p=.0045) and D30 (p=.0028), except for D7 when CAR-T cells began to expand (p=.9815), this demonstrated that steroids did not suppress the proliferation of CAR-T cells in PB. Secondly, the percentages of patients with detectable CAR-T cells in bone marrow (BM) and cerebrospinal fluid (CSF) were compared between steroid and non-steroid group, there were no differences both in BM (85.2% vs 78.6%, p=.923) and in CSF (68.6% vs 57.9%, p=.433), which implied steroids did not influence the trafficking of T-cells to BM and CSF. Thirdly, we monitored B-cell aplasia (BCA) in part of patients followed-up more than 2 months without further treatments, the percentages of patients with BCA in steroid group had no significant differences compared to non-steroid group at 2-month (p=.086) and 3-month (p=.146). Later, although limited cases left, in the steroid group, 100% of patients (4-month, 7/7; 5-month, 7/7; 6-month, 5/5) still maintained BCA and CR, indicating that corticosteroids did not impact the duration of functional CAR-T cells. In conclusion, corticosteroids do not compromise the treatment efficacy and kinetics of CAR-T cells, could be as a feasible and effective approach to manage CAR-T associated CRS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 810-810 ◽  
Author(s):  
Haiying Qin ◽  
Sang M Nguyen ◽  
Sneha Ramakrishna ◽  
Samiksha Tarun ◽  
Lila Yang ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Chain ◽  
Dual Targeting ◽  
Single Antigen ◽  
Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

scholarly journals The expression of CD26 and CD40 ligand is mutually exclusive in human T- cell non-Hodgkin's lymphomas/leukemias

Blood ◽  
1995 ◽  
Vol 86 (12) ◽  
pp. 4617-4626 ◽  
Author(s):  
A Carbone ◽  
A Gloghini ◽  
V Zagonel ◽  
D Aldinucci ◽  
V Gattei ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Mycosis Fungoides ◽  
Cell Lines ◽  
Critical Role ◽  
Cd40 Ligand ◽  
T Cell Lymphomas ◽  
Human T Cell ◽  
Hodgkin's Lymphomas

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T- cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

Cultured Human B Cell APCs Expanded Using Il-4 and CD40 Ligand Preferentially Promote a Type 2 T Cell Response to Alloimmune Stimulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2668-2668
Author(s):  
Abdul Tawab ◽  
Yoshiyuki Takahashi ◽  
Childs Richard ◽  
Kurlander J. Roger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cd40 Ligand ◽  
T Cell Response ◽  
Ifn Γ

Abstract In vitro stimulation of human peripheral blood B cells with recombinant IL-4 and CD40 ligand (CD40L) markedly increases their expression of MHC and costimulatory molecules, thus enhancing antigenic peptide presentation to T cells. Because these cells proliferate extensively in vitro (unlike monocytes or dendritic cells), they represent a promising and convenient reagent for the generation and maintenance of antigen-specific T cells for use in a variety of experimental or therapeutic settings. However, the impact of this type of B cell APC on cytokine production by responder T cells has hitherto not been examined. To address this issue, we stimulated normal human T cells with either allogeneic B cells (generated in vitro) or with MNCs obtained from the same donor. After 7 days, T cells were washed and re-challenged with the same APCs. The resulting alloreactive cytokine response was measured using quantitative ELISPOT methods and expressed as the frequencies of IFN-γ, IL-4, and IL-5 producing cells per thousand responder cells added. B cell- and MNC-primed cell lines both produced vigorous lymphokine responses, but B cell-stimulated T cells consistently produced more IL-5 spots (mean of 265 vs. 98/1000 responders, p<0.002) and fewer IFN-γ spots (163 vs 386/1000 cells, p<0.005) than MNC-stimulated cells. Further, the ratio of IFN-γ to IL-5 spots was almost ten-fold lower in B cell-stimulated cultures compared to MNC-induced cultures (0.67 vs. 5.2, p<0.001). ELISPOT studies assessing the ratio of IFN-γ to IL-4 spots and ELISA assays comparing IFN-γ and IL-5 levels from culture supernatants demonstrated the same pattern of marked type 2 skewing by B cells. This pattern was unaffected by the presence of anti-IL-4 antibody suggesting type 2 skewing was not mediated by IL-4. Cytokine skewing produced by B cells or MNC could be partially reversed by swapping MNC and B cells during re-stimulation on day 7, but this plasticity was markedly reduced after 3 (weekly) cycles of B cell or MNC re-stimulation in vitro. Type 2 skewing by B cells was enhanced when monocytes were removed from responder T cell populations by either depleting CD14+ positive cells or by positive selection of T cells prior to stimulation. In contrast, type 2 polarization could be prevented using recombinant IL-12. Not all cells of B-cell origin share the same propensity to type 2 skewing observed with IL-4/CD40L-stimulated B cells; under identical conditions, EBV-transformed B cells stimulated alloimmune T cells to produce a strong type 1 cytokine response comparable to that produced by MNCs. In summary, IL-4/CD40L-stimulated B cells strongly promote a type 2 T cell response during primary alloimmune challenge; this skewing can become fixed after repeated B cell stimulation. Investigators using these cells as APC should be aware of this potential phenomenon, particularly during primary T cell responses. It is also important to consider the factors described above that may exacerbate or ameliorate this effect.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

CLL Cells Can Diversify, Switch, and Differentiate in Response to Autologous T Cell Stimuli Present in a Murine Adoptive Transfer Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 315-315
Author(s):  
Piers E.M. Patten ◽  
Shih-Shih Chen ◽  
Davide Bagnara ◽  
Rita Simone ◽  
Sonia Marsilio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Adoptive Transfer ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Transfer Model ◽  
B Cell Differentiation ◽  
Cell Numbers ◽  
Nsg Mice

Abstract Abstract 315 Adoptive transfer of primary patient CLL cells into NOD/SCID/γcnull(NSG) mice results in engraftment and proliferation of CLL cells if autologous T cells are present. Formation of splenic follicles consisting of B cells interspersed and surrounded by T cells indicates engraftment. However, ultimately these CD20+ cells are lost several weeks later. We describe one of the mechanisms for this apparent loss: differentiation to plasma cells. Peripheral blood cells from 9 IgM+ CLL patients (6 U-CLL and 3 M-CLL) were adoptively transferred into NSG mice with enriched autologous CD3+ cells pre-activated with anti-CD3/28 beads. B and T cell engraftment and subset distributions were analyzed for 47 mice by immunohistochemistry (IHC) and flow cytometry (FC) at the time of sacrifice. The earliest and latest times of assessment were 12 and 124 days, respectively, after CLL cell injection. In some cases, CLL cells were labeled with CFSE to track cell division. At sacrifice, 3 engraftment patterns were observed. Pattern 1 (observed up to day 56) showed small follicles of CD20+ cells with low-moderate numbers of surrounding T cells. Intensely positive CD38 cells were inconspicuous. FC showed CD19+CD5+ cells with no increase in CD38 and variable CFSE dilution indicating lower levels of proliferation. Pattern 2 (observed throughout the study period) showed much higher T and B cell numbers. CD20+ cells were interspersed with and surrounded by principally CD4+ cells which were activated and functional as indicated by expression of Ki-67, PD-1, CD57, and T cell derived cytokines IFNγ and IL5 in plasma. Follicles contained CD20 and cytoplasmic Ig+ (cIg+) cells that double stained for IRF-4 and Blimp-1, transcription factors required for B cell differentiation. While Bcl-6 staining in these cells was minimal or absent, follicles from all 9 patients contained activation-induced deaminase (AID)+ cells. Cells with dim IgM expression localized to follicles; however, cells with intense IgM, IgA, or IgG were present both within, surrounding, and outside follicles matched by similar CD38 staining. Smaller populations of CD138+ cells surrounded follicles and were interspersed throughout non-follicular splenic areas. FC showed a novel CD19+CD5-CFSE-CD38++ population containing a CD138+ subset. Pattern 3 (observed in a limited subset of cases not before day 63) had minimal CD20+ cells by IHC, but noticeable populations of cIg+CD38+ and CD138+ cells interspersed amongst plentiful T cells. Such cells corresponded with cells with plasma cell morphology. Confirmation that differentiated cells were from the patient clone was achieved in 3 ways. First, in FACS sorted CD19+CD5+ and CD19+CD5-38++ cells from a subset of pattern 2 cases, RT-PCR revealed that all fractions contained both IGHC unswitched and switched clones identical to those found in the patients. Second, cases with pattern 3 engraftment generated CLL clonal switched and unswitched cDNA sequences. Finally, adoptive transfer of highly purified CD5+CD19+ patient cells generated IRF-4+Blimp-1+CD138+ cells. The generation of switched cells from all 9 patients indicated functional AID. In one U- CLL case, ultra-deep sequencing on pre-transfer and post-transfer human cells taken from mouse spleen revealed a significant number of new IGHVDJ mutations in spleen-derived cells. Such mutations targeted nucleotides typical for AID's action. In conclusion, CLL cells can diversify, switch, and differentiate in NSG mice in response to autologous T cell signals. The extent of this maturation is a function of T cell numbers and activity and the duration of the experiment. Differentiation without significant Bcl-6 expression suggests that follicles in NSG mice are not recapitulating classic germinal center reactions, possibly giving clues to the origin of CLL. Several features of poor prognosis disease were demonstrated (e.g., increased CD38 and AID expression with the development of clonally related switched transcripts) that might mirror clinical disease features. AID expressed by CLL cells is fully functional as indicated by de novo somatic hypermutation and class switch recombination. Both U-CLL and M-CLL clones respond in a similar manner in this model, suggesting the importance of T– B cell interactions in all types of CLL. Finally, the demonstration that cells can differentiate when appropriately induced may lead to novel therapeutic options for CLL. Disclosures: No relevant conflicts of interest to declare.

From T-cell Engineering to CAR therapy: Progress and Prospects

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-23-SCI-23 ◽  
Author(s):  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Second Generation ◽  
Human Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Stress Test ◽  
Lymphocytic Leukemia ◽  
Immunodeficient Mice ◽  
Recombinant Receptors

The genetic engineering of T cells provides a means to rapidly generate anti-tumor T cells for any cancer patient. This approach is predicated on gene transfer technology that enables the expression of receptors for antigen and other gene products in primary T cells. Tumor targeting may be achieved through the transfer of a physiological receptor for antigen, which is known as the T cell receptor (TCR), or synthetic fusion receptors, which we grouped under the general term of chimeric antigen receptor (CAR). CARs are recombinant receptors for antigen, which, in a single molecule, redirect T cell specificity and eventually enhance anti-tumor potency. Functional augmentation is achieved through the design of second generation CARs, which not only redirect cytotoxicity, but also reprogram T cell function and longevity through their costimulatory properties. The combined activating and costimulatory domains incorporated in second-generation CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19 CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Two decades ago, we selected CD19 as the prime target for developing our CAR technology and provided the first proof-of-principle that CD19-targeted human peripheral blood T cells could eradicate a broad range of B cell malignancies in immunodeficient mice (Brentjens RJ, Riviere I, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. 2003;9(3):279-86). CD19 has since become the poster child for CAR therapies. Complete remissions have been reported from several centers in patients with non-Hodgkin lymphoma, chronic lymphocytic leukemia and, most dramatically, acute lymphoblastic leukemia. Two types of second generation CARs, utilizing either CD28 or 4-1BB as their costimulatory signaling components, have been used in ALL patients. Both have yielded dramatic outcomes, in adults as well as in children. Our data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, while 4-1BB-based CARs direct a gradual T cell accumulation that may eventually overcome lesser functional potency. These distinct kinetic features can be exploited to further develop CAR T cell therapies for a variety of cancers. We have now modeled CD19 CAR therapy for ALL in a "stress test", wherein we purposefully lower the infused T cell doses to challenge the CAR therapy. We have compared novel CAR designs intended to recruit both CD28 and 4-1BB signaling. These quantitative analyses reveal striking disparities that hinge on subtle variations in the structural design of CARs and co-expressed costimulatory molecules. Remarkably, we find that some of the most effective engineering strategies activate and sustain the recruitment of the IFNβ pathway through the induction of IRF7, while lowering the induction of exhaustion markers relative to second generation CARs activating either CD28 or 4-1BB alone. The field is thus poised to move beyond the CD28 vs 4-1BB debate, which will be rendered obsolete by the emergence of superior CAR designs that coopt the use of costimulatory ligands, cytokines and/or checkpoint blockade inhibitors. A new field of immunopharmacology is emerging. Disclosures Sadelain: Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents & Royalties: Licensed patents on CARs.

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