Distinct Patterns of Systemic Immune Dysregulation in Indolent B Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3780-3780
Author(s):  
Petros Christopoulos ◽  
Dietmar Pfeifer ◽  
Kilian Bartholomé ◽  
Marie Follo ◽  
Paul Fisch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immune Dysregulation ◽  
Cell Phenotype ◽  
Cd4 Cells ◽  
Th Cells

Abstract Mutual interactions of the neoplastic clone with the non-neoplastic immune system may influence immune function and the clinical behaviour of lymphoma. Individuals with immunodeficiency or autoimmune diseases have an increased risk for lymphoma development. The immune microenvironment appears to have a major influence on the prognosis of indolent lymphomas. Conversely, leukemic lymphomas may also cause immunodeficiency: In CLL, direct lymphoma-T cell interactions, which may occur ubiquitously, induce defects in T cell functions (Görgün et al., 2005). We demonstrate here a systemic perturbance of cellular immunity in a prospective study in patients with untreated de novo, limited-stage, non-leukemic indolent B cell lymphomas. Calibrated, quantitative flow cytometry showed a significant reduction of circulating T helper (TH) cells in follicular (FL; n=11; p<0.005) and extranodal marginal zone (eMZL; n=7; p<0.05) lymphomas compared to age-matched healthy persons. Naive TH cells were strongest reduced to 51% (p=0.002) in FL and 24% (p=0.002) in eMZL. Regulatory T cells (CD4loCD25hi; CD4+FoxP3+) were affected less (p=0.04). T cell receptor excision circles within CD4+ cells as assessed by quantitative PCR were not altered in lymphoma patients, indicating neither increased increased thymic output nor homeostatic T cell proliferation to compensate the contracted pool of naive T cells. The TH memory compartments, the global numbers and subsets of CD8+ T (TC) cells, NK, and NKT cells were normal. The peripheral lymphocyte composition was altered differently in early CLL (stage Binet A; leukocyte counts < 28/nl; n=9) with increased TH (p=0.04) and TC (p=0.0002) cells. No significant changes in lymphocyte subsets were noted in monoclonal gammopathy of unknown significance (MGUS; n=6). The functional T cell phenotype in vivo was altered in eMZL as indicated by four- and twofold increased HLA-DR+ TH (p<0.02) and TC (p=0.05) cells. This T cell activation may also explain an increased fraction of terminally differentiated (CD45RA+CD27−) TC cells (p<0.05). Qualitatively similar abnormalities were seen in FL, where activated TH cells were more frequent (p<0.005), and in CLL, where activated TC cells were increased (p=0.04), but not in MGUS. Finally, an increased T cell activation may effect senescence, which was evident by elevated fractions of CD57+ and CD28− cells within the TC compartment of FL/eMZL (p<0.05) and CLL (p<0.005) patients. The activated T cell phenotype was paralleled by increased upregulation of activation markers (CD25, OX40, CD95, p<0.005 for each) and proliferation (p<0.005) by purified CD4 cells from FL/eMZL patients in a standardized anti-CD3/anti-CD28 stimulation culture. None of these parameters was significantly aberrant in CLL. Expression of the activation marker CD69, which is downregulated rapidly after T cell activation, was markedly reduced both in vivo and after in vitro stimulation in FL/eMZL. Collectively, these data demonstrate a global, “preactivated” and presenescent state of peripheral T cells in non-leukemic, indolent T cell lymphomas. Finally, a shift towards TH2 cells was evident in FL/eMZL TH stimulation cultures by increased secretion of IL-4 and IL-5 (p=0.01), but not of IL-2, IFNg, IL-10, and TNFa. This cytokine pattern was absent in CLL and MGUS. The TH2 shift, and the qualitative difference in the immune status in FL/eMZL versus CLL was validated by gene expression profiling of stimulated TH cells with Affymetrix U133 arrays. KEGG annotation revealed decreased expression of proximal TCR signalling molecules and TCR/CD28 transduction pathways with the exception of NFAT in FL/eMZL and CLL. Extensive correlative analyses between gene expression profiles and functional data indicated at least two distinct immune dysregulation patterns: A hyperreactivity/TH2 pattern which is operational even in early disease; and a B cell burden-dependent impairment of TCR signalling. The latter pattern predominates in CLL, which has a comparatively high B cell burden in early disease. These data are clinically relevant since we demonstrate in a prospective trial that untreated FL/eMZL patients fail to respond to protective hepatitis B vaccination (p<0.005). Precise definition of functional T cell defects will permit to study the causes, the prognostic influence, and potential reversibility of immune dysregulation patterns in indolent B cell lymphomas.

scholarly journals A Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills Lymphoma Cells with Minimal Cytokine Secretion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1671-1671
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Brian Avanzino ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Nog Mice

Abstract Introduction Along with CD20 and CD22, the restricted expression of CD19 to the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates specific to CD19 have been described, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-Ts) have been approved to treat leukemia. To date, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to CD19 circumvent the latter problem but to date have shown similar T-cell over-activation, as well as significant neurotoxicity. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that uses in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a selectively-activating αCD3 that induced potent T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we compared the low-activating (and Fc-containing) CD19 x CD3 to two pan T-cell activating bispecific CD19 x CD3 antibodies (blinatumomab and another developed in-house) in vitro and in vivo for T-cell activation, efficacy in killing lymphoma cells, and toxicity. Methods T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, Ramos, and Nalm6) was measured via calcein release in vitro. In vivo, NOG mice were engrafted with human peripheral blood mononuclear cells (huPBMC) and human lymphoma cell lines, and the mice treated with weekly injections of T-BsAbs. Tumor burden was evaluated via caliper measurement. Pharmacokinetic (PK) studies were performed in NOG mice using ELISA. Results EC50s for cytotoxicity were in the single-digit nanomolar range for the selective T cell activating T-BsAb and sub-nanomolar for the pan T-cell activating controls. The selective T cell activator showed markedly reduced cytokine release for all cytokines tested compared to the pan T-cell controls even at saturating concentrations. In vivo, established CD19 positive B-cell tumors were cleared in NOG mice in the presence of huPBMC. PK profiles of both molecules generated in-house (selective and pan T-cell activators) were consistent with those of an IgG in mice. No activation of T-cells was observed in vitro or in vivo in the absence of CD19 expressing target cells. Conclusions Both the selectively-activating and the pan T-cell activating control bispecific antibodies killed lymphoma cells in vitro and in vivo in a CD19-dependent manner. While the pan T-cell activating controls showed T-cell activation comparable to other CD3-engaging bispecifics, the selective activator induced significantly reduced cytokine secretion by T-cells and demonstrated a half-life consistent with other IgG antibodies. In summary, our selectively activating CD19 x CD3 T-BsAb shows promise as a lymphoma therapeutic differentiated from current T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment. Buelow:Teneobio Inc.: Employment. Avanzino:Teneobio, Inc.: Employment. Balasubramani:Teneobio, Inc.: Employment. Boudreau:Teneobio, Inc.: Employment. Clarke:Teneobio, Inc.: Employment. Dang:Teneobio, Inc.: Employment. Davison:Teneobio, Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Harris:Teneobio, Inc.: Employment. Jorgensen:Teneobio, Inc.: Employment. Li:Teneobio, Inc.: Employment. Medlari:Teneobio, Inc.: Employment. Narayan:Teneobio, Inc.: Employment. Ogana:Teneobio, Inc.: Employment. Pham:Teneobio Inc.: Employment. Prabhakar:Teneobio, Inc.: Employment. Rangaswamy:Teneobio, Inc.: Employment. Sankaran:Teneobio, Inc.: Employment. Schellenberger:Teneobio, Inc.: Employment. Ugamraj:Teneobio, Inc.: Employment. Trinklein:Teneobio, Inc.: Employment. Van Schooten:Teneobio, Inc.: Employment.

Reprogramming Leukemia Cells into Antigen Presenting Cells As a Novel Cancer Vaccination Immunotherapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3217-3217
Author(s):  
Miles Hamilton Linde ◽  
Christopher G Dove ◽  
Sarah F Gurev ◽  
Paul Phan ◽  
Feifei Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Myeloid Lineage ◽  
B Cell Maturation ◽  
Antigen Presenting ◽  
Tumor Eradication

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in B-cell maturation arrest and malignant transformation. Even with the addition of targeted therapies to conventional treatment regimens, prognosis for adults with high risk disease remains poor, particularly for those patients with relapsed or refractory disease. Despite an arrest in B cell maturation, we previously showed that human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage (McClellan et al, PNAS 2015). While the concept of forced differentiation was proposed several decades ago, no differentiation therapies have been effective in the treatment of B-ALL. Thus, we sought to investigate the therapeutic implications of myeloid lineage reprogramming of B-ALL cells. We speculated that myeloid-reprogramming of B-ALL cells into antigen presenting cells (APCs) could induce tumor-specific T cell responses through effective presentation of aberrant tumor-associated self-peptides. To test this hypothesis, we generated murine models of B-ALL capable of reprogramming to the myeloid lineage through the inducible expression of two transcription factors, CEBPα and PU.1. Ectopic expression of these factors efficiently reprogrammed B-ALL cells into myeloid-lineage APCs, expressing myeloid markers (CD11b, CD14, CD115, and Ly6C). Reprogramming ablated the tumorigenicity of these cells as they acquired APC characteristics, including phagocytic activity and expression of antigen presentation and co-stimulation molecules: MHC-I (3.13-fold, p=0.0018), MHC-II (8.6-fold, p<0.0001), CD80 (62.1-fold, p<0.0001), CD86 (107.6-fold, p<0.0001), and CD40 (92-fold, p<0.0001). Using chicken ovalbumin as a model antigen and DO11.10 transgenic CD4+ T cells, we demonstrated that reprogrammed B-ALL cells, but not parental blasts, can process and present both endogenous and exogenous peptides for antigen-specific T cell activation. To explore the therapeutic potential of B-ALL reprogramming, we engrafted immunodeficient (NSG) and immunocompetent syngeneic (BALB/c) mice with our B-ALL model and induced myeloid reprogramming in vivo. While B-ALL reprogramming in immunodeficient mice led to a three day extension in median survival (p=0.0016, n= 5 per group), all of the mice succumbed to their disease. Strikingly, B-ALL reprogramming in immunocompetent mice led to complete tumor regression and survival of the entire cohort 100 days post treatment (p<0.0001, n=10 per group), suggesting that reprogramming induced immune-mediated tumor eradication. Importantly, these animals were not susceptible to subsequent B-ALL re-challenge, demonstrating successful generation of durable, systemic, and protective immunity. In order to investigate the mechanism underlying tumor eradication, we depleted BALB/c mice of CD4+ or CD8+ T cells. Depletion of either T cell population abrogated the therapeutic benefit of B-ALL reprogramming, indicating that reprogrammed B-ALL cells stimulate T cell activation in vivo. Further analysis of the CD8 T cell repertoire by TCRVb chain usage revealed significant 10.3-fold (p=0.0109, n=5 per group) expansion of a single TCRVb chain family in response to B-ALL reprogramming, consistent with an oligoclonal T cell response. Following reprogramming, a 4.01-fold increase in the frequency of infiltrating T cells is observed in the bone marrow (p=0.0028), including both activated (CD25+/CD69+) (1.62-fold, p=0.018) and effector memory (CD44+CD62L-) (1.99-fold, p=0.0097) T cells. Finally, using a dual tumor model, we demonstrated that myeloid reprogramming-dependent T cell activation eradicates malignant cells systemically, as demonstrated by regression of contralateral tumors lacking reprogramming. Together, our data suggests that (1) B-ALL cells reprogrammed to the myeloid lineage can operate as potent APCs capable of presenting both endogenous and exogenous tumor-associated antigens, (2) in vivo B-ALL reprogramming elicits robust immune activation, dependent on both CD4+ and CD8+ T cells, and (3) B-ALL reprogramming-induced immune activation is potent, durable, tumor-eradicating, and systemic. Thus, reprogramming of B-ALL cells into APCs represents a novel immunotherapeutic strategy with potential clinical benefit for the management of B-ALL disease progression. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.

A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3721-3721
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Cells ◽  
Cytotoxicity Assays

Abstract Abstract 3721 Background: CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy. In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions: In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment.

Rituximab Directly Decreases T Cell Activation, Both In Vivo and In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3935-3935 ◽  
Author(s):  
Tamar Katz ◽  
Dina Stroopinsky ◽  
Jacob M. Rowe ◽  
Irit Avivi
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Cell Function ◽  
Activation Profile

Abstract Abstract 3935 Rituximab, a chimeric anti-C20 monoclonal antibody, has been extensively used over the last decade for the therapy of B cell malignancies. Recent clinical data suggest that rituximab may affect T cell function, increasing the risk of T cell dependent infections in heavily-treated patients. The current study was designed to investigate the effect of rituximab on T cell activation and assess T cell function following the addition of rituximab to purified T cells. The T cell activation profile, dependent on rituximab administration, was evaluated in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) generated from B-cell non-Hodgkin lymphoma (NHL) patients prior and immediately after the administration of 375 mg/m2 rituximab, were examined for the expression of inflammatory cytokines. The in vitro studies were performed by using CD25 depleted PBMCs or B cell depleted T cells (CD3+CD25-CD19-). The obtained cells were stimulated with allogeneic dendritic cells (DCs), in the absence or presence or 2 mg/ml rituximab. T cell activation was evaluated using immunophenotypic markers, cytokine profile and T cell proliferation assay. Eight NHL patients participated in the study. The level of T cells expressing inflammatory cytokines was significantly decreased following the administration of a single dose of rituximab. T cells expressing IL-2 declined from a mean level of 26.5% to 11.5% and the level of IFN- γ decreased from 22% to 4.2%. Further administration of rituximab, up to 4 weekly doses, resulted in an additional decline in the amount of inflammatory cytokine producing T cells to a level of 1.4% for IL-2 and 3.5% for IFN-g. However, repeated evaluation, performed at 4 months after completing rituximab, showed restoration of the inflammatory population. In accord with this inhibitory effect, in vitro stimulation of T cells with allogeneic DCs, in the presence of rituximab, resulted in a significant decrease in activation markers (CD25, GITR and CTLA-4) (Table 1). These changes were accompanied by a marked reduction in inflammatory cytokine production and proliferative capacity. Of interest, these inhibitory effects were also obtained whilst using B cell depleted T cells (CD3+CD25-CD19-). In conclusion, rituximab administration results in a transient T cell inactivation, demonstrated through the reduction in inflammatory cytokine production and T cell proliferation capacity. This effect appears to be non-B cell dependent, being obtained in the absence of B cell in the culture, and may account for clinical observations in ameliorating T-cell dependent disorders, such as graft-versus-host disease. Table 1. Activation profile depending on rituximab (in vitro) Without rituximab With rituximab *Activation marker (%) CD25 27 9 GITR 15.6 4.7 CTLA4 17.7 7 *Cytokines expression (%) IL-2 22 2 IL12 16 4 IFN-gamma 21 1.8 T cells proliferation (O.D.) DC stimulation 1.528 0.580 CMV stimulation 1.563 0.570 anti CD3/CD28 stimulation 0.705 0.407 * Gated out of lymphocytes Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mouse CD19-Targeted Chimeric Antigen Receptors That Include a 41BB Co-Stimulatory Domain Induce NFkB Signaling to Enhance T Cell Proliferation, Viability, and Leukemia Killing

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 843-843
Author(s):  
Gongbo Li ◽  
Nolan Beatty ◽  
Paresh Vishwasrao ◽  
Justin C. Boucher ◽  
Bin Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Car T Cells ◽  
Apoptosis Protein ◽  
Car T

Abstract CD19 targeted 2nd generation chimeric antigen receptor T (CAR T) cells have been successful against relapsed and/or refractory B cell malignancies. The pending FDA-approval of 2 separate CD19 targeted CAR T products highlight the need to understand the biology behind this novel therapy. CAR design includes a single-chain variable fragment, which encodes antigen-binding, fused to a transmembrane domain, co-stimulatory domain, and CD3ζ activation domain. The two CARs likely to be approved as standard of care include a 41BB or CD28 co-stimulatory domain. CD28 is a critical co-stimulatory receptor required for full T cell activation and persistence, while 4-1BB is a member of the tumor necrosis factor receptor family and also a critical T cell co-stimulatory factor. Early evaluation of the co-stimulatory domains role in CAR design confirmed that they are required to enhance T cell function, but lacked insight regarding their mechanism for this enhancement. Furthermore, clinical outcomes suggest that the co-stimulatory domains in CARs support different T cell functions in patients. For example, while overall outcomes are similar between 41BB (19BBz) and CD28-containing CARs (1928z), 19BBz CAR T cells can persist for years in patients, but functional 1928z CAR T cells rarely persist longer than a month. Recent studies are providing insight to these differences and have demonstrated that 4-1BB-containing CARs reduce T cell exhaustion, enhance persistence, and increase central memory differentiation and mitochondrial biogenesis, while CD28-containing CARs support robust T cell activation and exhaustion, and are associated with effector-like differentiation. However, these studies have been performed mostly in vitro or in immune deficient mice, which limits their ability to model complex immune biology. Therefore, we evaluated murine CD19-targeting CARs with a 4-1BB (m19BBz) or CD28- (m1928z) co-stimulatory domain in relevant animal models of immunity. We directly compared m19BBz and m1928z CAR T cell immune phenotype, cytotoxicity, cytokine production, gene expression, intracellular signaling, and in vivo persistence, expansion, and B cell acute lymphoblastic leukemia (B-ALL) eradication. In vitro assays revealed that m1928z CAR T cells had enhanced cytotoxicity and cytokine production compared to m19BBz CAR T cells. Also, evaluation of m1928z and m19BBz CAR T cells displayed similar immune phenotypes, but markedly different gene expression with m1928z CAR T cells upregulating genes related to effector function and exhaustion, while m19BBz CAR upregulated genes critical for NFkB regulation, T cell quiescence and memory. In vivo, both m1928z and m19BBz CAR T cells supported equivalent protection against B-ALL. Similar to patients, in our mouse models there are functional differences between the mouse CD19-targeted CAR T cells. At 1 week post-infusion m19BBz CAR T cells are present in the blood of mice at significantly greater levels than m1928z CAR T cells. Furthermore, m19BBz CAR T cells enhance proliferation and/or anti-apoptosis protein expression to enhance B cell killing, which is evidenced by our observation that irradiation significantly weakens the in vivo efficacy of m19BBz but not m1928z CAR T cells. Our results suggest that B cell killing by m1928z CAR T cells is not impacted by irradiation because of their efficacious cytotoxicity of B cells. In contrast, m19BBz CAR T cells have enhanced viability and anti-apoptosis protein expression, which allows them to compensate for reduced effector function. We investigated potential mechanisms for the enhanced viability and anti-apoptosis of m19BBz CAR T cells and determined that NFkB signaling is upregulated much greater by m19BBz than m1928z. We have observed this difference in both a reporter cell line and primary mouse T cells. We are now dissecting what cellular components mediate increased NFkB signaling by the m19BBz CAR. Our animal models recapitulate equivalent anti-leukemia efficacy of CD19-targeted CAR T cells regardless of co-stimulatory domain, but underscore that anti-leukemia killing is mediated by different methods depending on the co-stimulatory domain. Our work sheds light on how 4-1BB mechanistically regulates and impacts CAR T function and has implications for future CAR design and evaluation. Disclosures No relevant conflicts of interest to declare.

Rap1-GTP Promotes the Generation of Regulatory T Cells in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 110-110
Author(s):  
Lequn Li ◽  
Rebecca Greenwald ◽  
Esther M. Lafuente ◽  
Dimitrios Tzachanis ◽  
Alla Berezovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd4 Cells

Abstract Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

scholarly journals Bifunctional iRGD-anti-CD3 enhances antitumor potency of T cells by facilitating tumor infiltration and T-cell activation

2021 ◽  
Vol 9 (5) ◽  
pp. e001925
Author(s):  
Shujuan Zhou ◽  
Fanyan Meng ◽  
Shiyao Du ◽  
Hanqing Qian ◽  
Naiqing Ding ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Confocal Microscope ◽  
Mechanistic Studies ◽  
Tumor Spheroids ◽  
Antitumor Effects ◽  
Transport Pathway

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

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