Tumor-Targeted Human CD4+ CD25hi Regulatory T Cells Effectively Inhibit T Cell –mediated Rejection of CD19+ Tumors in An in Vivo xenotransplant Tumor Model

Abstract The genetic targeting of human T cells to selected tumor antigens offers a novel means to investigate human immunobiology and treat cancer. T cells may be genetically modified to target specific antigens through the introduction of genes encoding chimeric antigen receptors (CARs). We have previously demonstrated that human T cells targeted in this manner to the CD19 antigen, expressed on normal B cells as well as most B cell tumors, eradicate systemic human CD19+ B cell malignancies in SCID-Beige mice. However, in the clinical setting, the anti-tumor efficacy of these T cells may be impaired by endogenous suppressive elements of the host immune system, including CD4+ CD25hi Foxp3+ regulatory T cells (Tregs). Significantly, Tregs are often increased in the blood and infiltrate the tumor of cancer patients which has been correlated with poor patient outcome and ineffective anti-tumor immunity. In order to study the in vivo impact of Tregs on adoptive therapy with CD19 targeted effector T cells, we developed a murine model wherein human Tregs, similarly targeted to the tumor, are infused prior to adoptive transfer of targeted cytotoxic T cells. To do so, we initially isolated natural Tregs from healthy donor peripheral blood mononuclear cells. Isolated Tregs were subsequently modified to express CARs through retroviral gene transfer. Subsequently, CAR+ Tregs were rapidly expanded either by activation on NIH-3T3 fibroblasts modified to express CD19 and the CD80 costimulatory ligand (3T3(CD19/CD80)), or non-specifically using CD3/CD28 antibodycoated magnetic beads. Expanded CAR+ Tregs exhibited potent suppressive function in vitro inhibiting both effector T cell proliferation as well as cytotoxicity. In vivo, CAR+ Tregs specifically traffic to established tumor in SCID-Beige mice. Significantly, injection of CD19-targeted Tregs into SCID-Beige mice bearing established human CD19+ tumors at 24 hours prior to infusion with CD19-targeted effector T cells, completely abrogated effector T cell function even at Treg:Teff ratios as low as 1:8. We further found that full suppression was dependant both on Treg localization to the tumor site as well as in vivo activation through the CAR. Finally, we show that a pre-conditioning regimen with low-dose cyclophosphamide, which failed to eradicate tumor, was able to reverse the CAR+ Treg mediated inhibition and restore the anti-tumor activity by the targeted effector T cells. In conclusion, we have developed a robust model ideally suited to the study of in vivo Treg-Teff interactions. Furthermore, the data generated from this model to date have significant implications with respect to the application of adoptive T cell therapies in the clinical setting. Namely, the presence of endogenous Tregs at the site of tumor is likely to significantly compromise the anti-tumor activity of adoptively transferred tumor targeted T cells. This inhibition may be reversed by preconditioning regimens designed to eradicate endogenous Tregs. The findings presented here should be considered in the design of future clinical trials utilizing T cell-based adoptive therapies of cancer.

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Novel Multi-Target Immunosuppressive Approach for Treatment of Severe Aplastic Anemia

Blood ◽

10.1182/blood.v126.23.3611.3611 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3611-3611 ◽

Cited By ~ 1

Author(s):

Kyle Blaine Russell ◽

Brandon P Theall ◽

Lisandra Hernandez ◽

Hannah M Wavering ◽

Vesna Jurecic ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

Aplastic Anemia ◽

Effector T Cells ◽

Prolonged Treatment ◽

Effector T Cell ◽

New Therapy

Abstract Aplastic Anemia (AA) is an immune-mediated form of acquired bone marrow failure (BMF), which is life-threatening in its severe form (SAA). Fundamental pathological features of AA include development and expansion of auto-reactive effector T cells, effector T cell-mediated apoptosis of all hematopoietic cells (including progenitors and hematopoietic stem cells (HSCs)), BM aplasia, pancytopenia, depletion of HSCs, and severe reduction and functional impairment of regulatory T cells (Tregs). Current standard treatments for AA include: (1) immuno-suppressive therapy (IST) with cyclosporine A (CyA) and anti-thymocyte globulin (ATG) which targets all T cells, and (2) allogeneic or matched unrelated donor BM transplant. While IST remains a standard treatment modality, it is not very effective in treating already ongoing and relapsed AA or SAA. There is really no effective therapy for patients with refractory and relapsed AA who are ineligible for BMT. Among different pathophysiological features of AA, IST targets only the effector T cells, and is much more effective in the early than later stages of AA. Moreover, the combination of IST with other immunosuppressive agents (mycophenolate mofetil, sirolimus etc.) or growth factors does not improve the response or survival of AA patients. Since the incidence of AA is on the rise, there is an urgent need for more efficient new therapies that can attenuate the progression and severity of AA in patients with refractory and relapsed AA who are waiting for or are not candidates for BMT. The complex immune and hematological pathophysiology of AA requires new multipurpose treatment approaches. Accumulating evidence shows that β2 integrin CD11b/CD18 (Mac-1) negatively regulates T cell responses and activation, attenuates inflammation, and facilitates the maintenance of tolerance to self-antigens. For example, activated Mac-1 significantly reduces the T cell-activating capacity of dendritic cells (DCs), represses DC cross-priming of cytotoxic T cells, negatively regulates NK cell activation and function, suppresses differentiation of Th17 T cells which are associated with AA and other autoimmune diseases, ameliorates experimental autoimmune hepatitis, and negatively regulates BCR signaling and maintains autoreactive B cell tolerance. For that reason, Mac-1 is an attractive molecular target for new immune-modulating therapies of autoimmune diseases. Using the clinically relevant mouse SAA model we have evaluated the therapeutic efficacy of Leukadherins (LA1-LA3), novel small molecule agonists and activators of Mac-1, as a novel multipurpose immunosuppressive and anti-inflammatory approach to treat AA. The present studies have demonstrated that administration of LA1 safely and significantly (1) suppresses expansion of effector T cells, (2) decreases effector T cell-mediated apoptosis of target BM cells, (2) reduces BM aplasia, (3) minimalizes the loss of HSCs and progenitors, and (4) attenuates the severity of SAA. Furthermore, prolonged treatment of developing SAA with LA1 has therapeutic effects since it not only attenuates the progression and severity of SAA, but also converts otherwise fatal SAA into a survivable disease in mouse SAA models. To begin to address mechanism for these findings we found that LA1 treatment significantly reduces the antigen presenting capacity and T cell activating capacity of DCs. Importantly, in vivo LA1 treatment significantly increases the population of regulatory T cells (Tregs) which may also contribute to the above effects of LA1 on SAA. Simultaneous targeting of multiple pathophysiological features of AA underscores the clinically relevant potential of LA1 treatment as a novel promising multi-target immunosuppressive therapy that can safely and efficiently attenuate the severity of AA and reduce the need for BMT. We are also further evaluating the potential of LA1 treatment combined with IST or in vivo Treg expansion approaches (low dose rIL-2 therapy) to safely and more effectively attenuate the progression and severity of AA in pre-clinical mouse SAA models. These studies will provide an important platform for further translational and clinical testing of LAs as: (1) New therapy to manage ongoing AA in patients who are not responding to IST and are not candidates for BMT, (2) New therapy for relapsed AA, and/or (3) Adjuvant therapy for AA patients who are undergoing IST and are awaiting BM transplant. Disclosures Levy: Allergan: Consultancy.

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Efficacy and Long Term Survival of Genetically Modified T Cells Targeting CD19 in a Syngeneic Immune Competent Transgenic Murine Tumor Model Is Dependent on Prior Lymphodepleting Chemotherapy.

Blood ◽

10.1182/blood.v110.11.2754.2754 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2754-2754

Author(s):

James Lee ◽

Yan Nikhamin ◽

Gavin Imperato ◽

Adam Cohen ◽

Michel Sadelain ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Clinical Setting ◽

Peripheral Blood ◽

Tumor Model ◽

B Cell Malignancies ◽

Car T Cells ◽

Human T Cells ◽

Car T

Abstract T cells may be genetically modified ex vivo to target specific antigens by retroviral transduction of genes encoding chimeric antigen receptors (CARs). We have previously constructed a CAR, termed 19z1, specific for the CD19 antigen expressed on most B cell malignancies. Human T cells modified to express the 19z1 CAR specifically eradicate systemic human CD19+ tumors in SCID-Beige mice. However, these models are limited by the xenogeneic nature of the human T cells and tumor cells and the immune compromised state of the host. Here, we studied the biology of adoptively transferred 19z1+ T cells in a syngeneic immune competent murine model designed to better mimic the clinical setting of patients with B cell malignancies. We utilized transgenic C57BL6 mice which lack expression of mouse CD19 (mCD19−/−) and have a single copy of the human CD19 (hCD19+/−) gene (C57BL6(mCD19−/− hCD19+/−)) kindly provided by Dr. T. Tedder, Duke University. These mice are functionally immune-competent with hCD19 expression restricted to the B cell population. To assess whether syngeneic 19z1+ T cells were capable of eradicating normal hCD19+ B cells, we infused C57BL6(mCD19−/− hCD19+/−) mice with either 19z1+ or control prostate specific membrane antigen-targeted (Pz1+) T cells. As assessed by flow cytometric analysis of peripheral blood, we neither found evidence of hCD19+ B cell aplasias in 19z1+ T cell treated mice nor were able to demonstrate the persistence of infused CAR+ T cells. To investigate whether the lack of 19z1+ T cell efficacy and persistence was due to an absence of homeostatic drive, we next lymphodepleted C57BL6(mCD19−/− hCD19+/−) mice with cyclophosphamide prior to T cell infusion. Mice lymphodepleted prior to 19z1+ T cell infusion demonstrated marked and sustained B cell aplasias when compared to lymphodepleted Pz1+ T cell and non-lymphodepleted T cell treated controls. Furthermore, while no CAR+ T cells were identifiable in the Pz1 and non-lymphodepleted control groups, 19z1+ T cells were consistently present in the peripheral blood of the cyclophosphamide pre-treated, 19z1+ T cell treated mice (3–5% of white blood cells). To assess the anti-tumor efficacy of the 19z1+ T cells, we next established a systemic tumor model utilizing mouse EL4 thymoma cells retrovirally modified to express hCD19 (EL4(hCD19)). C57BL6(mCD19−/− hCD19+/−) mice pre-treated with cyclophosphamide, subsequently infused systemically with EL4(hCD19) tumor, followed by systemic 19z1+ T cell infusion, had a significant survival advantage (80% survival at >120 days) over untreated controls or controls treated with Pz1+ T cells or 19z1+ T cells in the absence of lymphodepletion (0% survival). In conclusion, we have developed a syngeneic immune competent tumor model of hCD19 disease that is highly relevant to the clinical setting. Using this model, we demonstrate the significance of lymphodepletion on the prolonged in vivo persistence and anti-tumor efficacy of 19z1+ T cells. Data derived from this model will be correlated to findings obtained from a recently initiated clinical trial for patients with chronic lymphocytic leukemia, and will significantly impact the design of subsequent trials in the future.

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Juzentaihoto Exerts Anti-Allergic Effects by Inhibiting Effector T-Cell Activation and Inducing and/or Activating Regulatory T Cells in a Murine Model of Contact Hypersensitivity

International Archives of Allergy and Immunology ◽

10.1159/000518448 ◽

2021 ◽

pp. 1-13

Author(s):

Atsushi Tsuge ◽

Sho Yonekura ◽

Satomi Watanabe ◽

Yuta Kurosaki ◽

Shinsuke Hisaka ◽

...

Keyword(s):

T Cells ◽

Monoclonal Antibodies ◽

T Cell ◽

Regulatory T Cells ◽

T Cell Activation ◽

Cell Activation ◽

Effector T Cells ◽

Contact Hypersensitivity ◽

Dependent Manner ◽

Effector T Cell

Background: Juzentaihoto (JTT) is a Kampo prescription that has been used clinically for treating skin diseases such as atopic dermatitis in Japan. We have previously studied the anti-allergic effects of JTT on 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) in mice and demonstrated that it significantly suppresses ear swelling in a dose-dependent manner. However, the mechanism underlying the anti-allergic actions of JTT is obscure. Methods: We investigated the mechanism underlying the anti-allergic effects of JTT using a TNCB-induced murine CHS model and adoptive cell transfer experiments. Results: We showed that the anti-allergic effects of JTT are due to inhibition of effector T-cell activation and induction and/or activation of regulatory T cells. Furthermore, ex vivo experiments confirmed the effect of JTT on the activation of effector T cells and regulatory T cells, as interferon-γ production decreased, whereas interleukin (IL)-10 production increased, in the cultured lymphocytes obtained from 5% TNCB-sensitized mice treated with anti-CD3ε and anti-CD28 monoclonal antibodies. Flow cytometry showed that the CD4+CD25+Foxp3+, CD4+CD25+Foxp3−, and CD8+CD122+ cell population increased after oral administration of JTT. Finally, the anti-allergic effect of JTT by inducing and/or activating regulatory T cells (Tregs) was confirmed to be mediated by IL-10 through in vivo neutralization experiments with anti-IL-10 monoclonal antibodies. Conclusion: We suggested that JTT exerts anti-allergic effects by regulating the activation of effector T cells and Tregs involved in murine CHS model.

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Impact of Regulatory T Cells on Antigen Specific T Cell Response Using Recombinant Chimeric T Cell Receptors In Vivo.

Blood ◽

10.1182/blood.v108.11.5475.5475 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5475-5475

Author(s):

David M. Kofler ◽

Markus Chmielewski ◽

Heike Koehler ◽

Tobias Riet ◽

Patrick Schmidt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Tumor Cells ◽

T Cell Receptors ◽

Cell Response ◽

Effector T Cells ◽

T Cell Response ◽

Cell Receptors

Abstract Recombinant T cell receptors with defined specificity against tumor cells are a promising experimental approach in the elimination of residual leukemia and lymphoma cells. It is so far unresolved whether regulatory T cells with suppressor activities impair the efficiency of cytolytic T cells grafted with a recombinant immunoreceptor. The frequency of regulatory T cells is highly increased in tumor patients and their suppressive function seems to play a role in the fail of an autologous T cell response against the malignant cells. In this study we analyzed the antigen-triggered, specific activation of receptor grafted T cells in the presence or absence of regulatory CD4+CD25high T cells. CD3+ T cells were grafted with CEA-specific immunoreceptors containing the CD3-zeta signaling domain for T cell activation. Co-cultivation of receptor grafted effector T cells together with regulatory T cells repressed proliferation of the effector cells and decreased IL-2 secretion. Secretion of IFN-gamma and IL-10 was not impaired. Interestingly, the cytotoxicity of grafted effector T cells towards CEA-expressing tumor cells was not impaired by regulatory T cells in vitro. To evaluate the relevance in vivo, we used a Crl:CD1 Nu/Nu mouse model to assess growth of CEA+ tumor cells in the presence of receptor grafted effector T cells and of regulatory T cells. Mice inoculated with tumor cells together with CD3+ effector T cells without immunoreceptor and regulatory T cells developed earlier tumors with faster growth kinetics compared to mice that were inoculated with tumor cells, CD3+ T cells and CD4+CD25- control T cells. Using effector T cells that were equipped with a recombinant CEA-specific CD3-zeta immunoreceptor, 2 of 5 mice developed a tumor in the presence of regulatory T cells while none of the mice developed a tumor in the absence of regulatory T cells. Taken together, regulatory T cells obviously impair an antigen-specific, anti-tumor T cell attack in vivo. This seems to be due to repression of proliferation of the effector T cells and not to diminished cytotoxicity. These findings have major impact on the design of clinical studies involving adoptively transferred effector T cells.

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GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

Blood ◽

10.1182/blood.v120.21.1890.1890 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1890-1890

Author(s):

Shen Sylvie ◽

Ning Xu ◽

Guy Klamer ◽

Tracey O'Brien ◽

Alla Dolnikov

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Progenitor Cells ◽

Cell Development ◽

Memory T Cell ◽

T Cell Reconstitution ◽

Human T Cells

Abstract Abstract 1890 Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3'-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures: No relevant conflicts of interest to declare.

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Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry

Blood ◽

10.1182/blood.v86.4.1408.bloodjournal8641408 ◽

1995 ◽

Vol 86 (4) ◽

pp. 1408-1419 ◽

Cited By ~ 377

Author(s):

LJ Picker ◽

MK Singh ◽

Z Zdraveski ◽

JR Treer ◽

SL Waldrop ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Subset ◽

Human Memory ◽

Effector T Cells ◽

Accessory Cell ◽

Effector T Cell ◽

Cytokine Synthesis

The array of cytokines produced by T cells in effector sites is a primary means by which these cells mediate host defense. It is well recognized that cloned T cells are heterogeneous with regard to cytokine synthesis and, thus, in their ability to mediate specific immune responses, but the extent to which the patterns of cytokine secretion observed in cloned cells reflect actual populations of memory/effector T cells existing in vivo is largely unknown. Here, we report our findings using a multiparameter flow cytometric assay that allows simultaneous determination of an individual T-cell' ability to produce multiple cytokines and its phenotype after only short (4 to 8 hours) in vitro incubation with an activating stimulus and the secretion inhibitor Brefeldin A. This assay shows a rapid accumulation of interleukin-2 (IL-2), IL-4, and gamma-interferon (gamma-IFN) in the cytoplasm of CD4+ cells after stimulation with either accessory cell- independent (phorbol 12-myristate 13-acetate [PMA] + ionomycin [I]) or accessory cell-dependent (staphylococcal enterotoxins [SE] A and B) T- cell-activating stimuli. Further analysis showed that production of gamma-IFN and IL-4 is predominantly, if not exclusively, restricted to the CD45ROhigh memory/effector T-cell subset, whereas IL-2 may be produced by both the CD45ROhigh and CD45ROlow subsets. Simultaneous determination of IL-2 and gamma-IFN production among CD45ROhigh/CD4+ T cells showed distinct subsets that produce each of these cytokines alone (an average of 30% for IL-2 alone, 8% for gamma-IFN alone), both (16%), or neither (46%). Similar analyses with the small IL-4-producing memory/effector T-cell subset (only 4.3% of total CD4+/CD45ROhigh T cells) showed that an average of 51% of these IL-4-producing cells also synthesize average of 51% of these IL-4-producing cells also synthesize IL-2, 23% synthesize only IL-4, 16% synthesize all three cytokines, and 9.6% synthesize IL-4 and gamma-IFN. These patterns of cytokine synthesis were found to be similar with both PMA + I and SEA/SEB stimulation and were observed in both peripheral blood memory/effector CD4+ T cells and in T cells of similar phenotype obtained from cutaneous delayed-type hypersensitivity sites. Taken together, these data strongly support the in vivo existence of human memory/effector T- cell subsets with “preprogrammed” cytokine synthesis potential, although they suggest that these subsets may be more complex than originally proposed in the TH1/TH2 hypothesis.

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Regulatory T-cells and cAMP suppress effector T-cells independently of PKA–CREM/ICER: a potential role for Epac

Biochemical Journal ◽

10.1042/bj20130064 ◽

2013 ◽

Vol 456 (3) ◽

pp. 463-473 ◽

Cited By ~ 23

Author(s):

Amanda G. Vang ◽

William Housley ◽

Hongli Dong ◽

Chaitali Basole ◽

Shlomo Z. Ben-Sasson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Regulatory T Cell ◽

Potential Role ◽

Cell Function ◽

Effector T Cells ◽

Immune Control ◽

Effector T Cell ◽

Camp Signalling

Immune control of effector T-cell function can be mediated by cAMP signalling and regulatory T-cell action independently of the PKA–CREM/ICER signalling pathway. EPAC may act as an alternative cAMP effector in this process.

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Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Blood ◽

10.1182/blood-2021-151598 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2406-2406

Author(s):

Giorgia Zanetti ◽

Giuseppina Astone ◽

Luca Cappelli ◽

William Chiu ◽

Maria Teresa Cacciapuoti ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Ex Vivo ◽

Human Immune System ◽

Human T Cells ◽

Human Immune

Abstract Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

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The Non-Signaling Extracellular Spacer Domain of CD19-Specific Chimeric Antigen Receptors Is Decisive for in Vivo Anti-Tumor Activity

Blood ◽

10.1182/blood.v120.21.951.951 ◽

2012 ◽

Vol 120 (21) ◽

pp. 951-951 ◽

Cited By ~ 2

Author(s):

Michael Hudecek ◽

Anne Silva ◽

Paula L. Kosasih ◽

Yvonne Y. Chen ◽

Cameron J. Turtle ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Target Cells ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Car T ◽

Anti Tumor Activity

Abstract Abstract 951 Adoptive immunotherapy with T cells engineered by gene transfer to express CD19-specific chimeric antigen receptors (CARs) has the potential to induce remissions in patients with advanced B cell malignancies. CARs are synthetic receptors with an extracellular antigen-binding domain (scFv), a spacer domain that provides separation of the scFv from the cell membrane and an intracellular signaling module, most commonly the CD3ζ chain and one or more costimulatory domains such as CD28 or 4-1BB. Several clinical trials with CD19-CAR T cells in small cohorts of patients with B cell tumors have been reported with variable results. Although most studies have used the CD19-specific FMC63 scFv as the tumor-targeting moiety, the extracellular, transmembrane and intracellular CAR domains used in each trial have been distinct, and an emerging paradigm is that including costimulation in the design of the CAR is key to achieving anti-tumor activity in vivo. In this study, we analyzed the influence of extracellular spacer domain length on the in vitro and in vivo function of CD19-CARs. We constructed a panel of four CD19-CARs comprised of the FMC63 scFv and either a long spacer derived from the IgG4-Fc Hinge-CH2-CH3 domain (229 AA) or a short Hinge domain only spacer (12 AA). Each CAR contained a signaling module of CD3ζ with CD28 (short/CD28; long/CD28) or 4-1BB (short/4-1BB; long/4-1BB). We transduced CD8+ CD45RO+ CD62L+central memory T cells of normal donors with each of the CARs, enriched transduced T cells to >90% purity by immunomagnetic selection using a tEGFR marker encoded in the CAR vector, and expanded CAR transduced T cells using a uniform culture protocol. We compared the in vitro function of T cell lines expressing each of the CD19-CARs and confirmed specific cytolytic activity against CD19+ target cells including K562/CD19, and Raji and JeKo-1 lymphoma cells. Quantitative cytokine analyses showed higher levels of IFN-γ, TNF-α, IL-2 production in T cells expressing CD19-CARs with CD28 costimulatory domain compared to the corresponding constructs with 4-1BB, consistent with prior work. T cells expressing each of the CD19-CARs proliferated in vitro after stimulation with K562/CD19 and Raji tumor cells by CFSE dye dilution, with the strongest proliferation observed in T cells expressing the CD19-CAR ‘long/CD28’, consistent with the highest levels of IL-2 production by T cells expressing this construct. We then analyzed the in vivo anti-tumor efficacy of each CD19-CAR in immunodeficient NOD/SCID/g−/− (NSG) mice engrafted with firefly luciferase transduced Raji cells. Tumor was inoculated on day 0, and once tumor was established (day 7), the mice received a single dose of 2.5×106̂ T cells expressing each CD19-CAR, a tEGFR control vector, or were left untreated. Surprisingly, only T cells expressing CD19-CARs with a short spacer domain (short/CD28 and short/4-1BB) eradicated the Raji tumors and led to long-term tumor-free survival of all mice. T cells expressing CD19-CARs with a long spacer domain (long/CD28 and long/4-1BB) did not confer a significant anti-tumor effect and all mice expired from systemic lymphoma at a similar time as control and untreated mice. The anti-tumor efficacy in vivo of T cells modified with long spacer CD19-CARs could not be improved by increasing CAR T cell dose 4 fold, or by including additional costimulatory domains into the CD19-CAR (long/CD28:4-1BB). Serial analyses in peripheral blood, bone marrow and spleen showed dramatically lower numbers of transferred T cells in mice treated with long spacer CD19-CARs compared to mice treated with short spacer CD19-CARs or control T cells. Further analysis revealed that despite strong activation in vivo as assessed by upregulation of CD69 and CD25, CD19-CARs with long extracellular spacer domain induced a high rate of activation induced T cell death in vivo. Collectively, these results demonstrate that the extracellular spacer domain that lacks intrinsic signaling function is critical in the design of effective CD19-CARs, and illustrates that tailoring spacer length is likely to be essential for designing effective CARs specific for other tumor antigens. Disclosures: No relevant conflicts of interest to declare.

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In vivo sites and cellular mechanisms of T reg cell–mediated suppression

Journal of Experimental Medicine ◽

10.1084/jem.20060214 ◽

2006 ◽

Vol 203 (3) ◽

pp. 489-492 ◽

Cited By ~ 78

Author(s):

Alexander Y. Rudensky ◽

Daniel J. Campbell

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

T Cell Activation ◽

Cell Activation ◽

Effector T Cells ◽

Cell Contact ◽

Cellular Mechanisms ◽

Effector T Cell

Regulatory CD4 T (T reg) cells control immune responses to self-antigens and pathogens. However, where T reg cells act to curtail effector T cells in vivo and what stage of effector T cell activation or differentiation is targeted by T reg cells remain unknown. Furthermore, a requirement for direct effector T cell–T reg cell contact in vivo has not been ascertained. Varying answers to these important questions have been provided by several new studies.

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