Combining PD-1 blockade with T-cell redirecting bispecific antibodies for solid cancer therapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 98-98
Author(s):  
Chien-Hsing Chang ◽  
Yang Wang ◽  
Diane L Rossi ◽  
Rongxiu Li ◽  
Edmund A. Rossi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Scid Mice ◽  
Bispecific Antibodies ◽  
Human Pbmcs ◽  
In Vivo Models ◽  
3D Spheroids

98 Background: Bispecific antibodies (bsAbs) for redirecting T cells to cancers have shown promise in both preclinical and clinical studies. However, clinical results have been disappointing in solid cancers. We have applied the DOCK-AND-LOCK method to generate a novel class of trivalent bsAbs, each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. Herein we report the characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2- and CEACAM5-expressing cancer cells, respectively. Methods: Human breast and colonic cancer cell lines were grown in monolayer cultures or as 3D spheroids for in vitro evaluation. NOD/SCID mice carrying xenografts of MDA-MB-231 (a TNBC line constitutively expressing Trop-2 and PD-L1) were used for in vivo studies. A human PD-1 antagonistic murine hybridoma antibody was subsequently converted to its chimeric form (IMMU-cPD-1). Human PBMCs, or T cells isolated from buffy coats by negative selection, were used as effector cells in cytotoxicity assays. The effect of IMMU-cPD-1 on cancer cells pretreated with IFN-γ to induce the expression of PD-L1 was compared with those not pretreated. Results: (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at low picomolar concentrations, with similar potency observed for drug-resistant cells. The antitumor efficacy was demonstrated for (E1)-3s plus human PBMCs in NOD/SCID mice bearing MDA-MB-231, and for human PBMCs combined with (E1)-3s or (14)-3s in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, with the addition of IMMU-cPD-1, the benefit of PD-1 blockade was indicated by increased cell death in 3D spheroids and longer survival of MDA-MB-231-bearing mice. Conclusions: These results highlight the potency of (E1)-3s and (14)-3s as T-cell redirecting bsAbs, emphasize the potential of combining such bsAbs with immune checkpoint inhibitors to improve the therapeutic activity in the immunotherapy of solid cancers, and provide a basis for using 3D spheroids as an alternative to in vivo models for evaluating T-cell functions.

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

scholarly journals TCR β chain–directed bispecific antibodies for the treatment of T cell cancers

2021 ◽  
Vol 13 (584) ◽  
pp. eabd3595 ◽  
Author(s):  
Suman Paul ◽  
Alexander H. Pearlman ◽  
Jacqueline Douglass ◽  
Brian J. Mog ◽  
Emily Han-Chung Hsiue ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Cancer ◽  
Bispecific Antibodies ◽  
Target Antigen ◽  
Human T Cell ◽  
Selective Targeting ◽  
Β Chain

Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen–targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR β chain generated from 1 of 30 TCR β chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.

scholarly journals Single-cell RNA sequencing reveals distinct cellular factors for response to immunotherapy targeting CD73 and PD-1 in colorectal cancer

2021 ◽  
Vol 9 (7) ◽  
pp. e002503
Author(s):  
Miok Kim ◽  
Yong Ki Min ◽  
Jinho Jang ◽  
Hyejin Park ◽  
Semin Lee ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Trials ◽  
T Cells ◽  
T Cell ◽  
In Vivo Models ◽  
Single Cell Rna Sequencing ◽  
Tcr Diversity ◽  
Cancer Immunotherapies

BackgroundAlthough cancer immunotherapy is one of the most effective advanced-stage cancer therapies, no clinically approved cancer immunotherapies currently exist for colorectal cancer (CRC). Recently, programmed cell death protein 1 (PD-1) blockade has exhibited clinical benefits according to ongoing clinical trials. However, ongoing clinical trials for cancer immunotherapies are focused on PD-1 signaling inhibitors such as pembrolizumab, nivolumab, and atezolizumab. In this study, we focused on revealing the distinct response mechanism for the potent CD73 ectoenzyme selective inhibitor AB680 as a promising drug candidate that functions by blocking tumorigenic ATP/adenosine signaling in comparison to current therapeutics that block PD-1 to assess the value of this drug as a novel immunotherapy for CRC.MethodsTo understand the distinct mechanism of AB680 in comparison to that of a neutralizing antibody against murine PD-1 used as a PD-1 blocker, we performed single-cell RNA sequencing of CD45+ tumor-infiltrating lymphocytes from untreated controls (n=3) and from AB680-treated (n=3) and PD-1-blockade-treated murine CRC in vivo models. We also used flow cytometry, Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) models, and in vitro functional assays to validate our new findings.ResultsWe initially observed that the expressions of Nt5e (a gene for CD73) and Entpd1 (a gene for CD39) affect T cell receptor (TCR) diversity and transcriptional profiles of T cells, thus suggesting their critical roles in T cell exhaustion within tumor. Importantly, PD-1 blockade significantly increased the TCR diversity of Entpd1-negative T cells and Pdcd1-positive T cells. Additionally, we determined that AB680 improved the anticancer functions of immunosuppressed cells such as Treg and exhausted T cells, while the PD-1 blocker quantitatively reduced Malat1high Treg and M2 macrophages. We also verified that PD-1 blockade induced Treg depletion in AOM/DSS CRC in vivo models, and we confirmed that AB680 treatment caused increased activation of CD8+ T cells using an in vitro T cell assay.ConclusionsThe intratumoral immunomodulation of CD73 inhibition is distinct from PD-1 inhibition and exhibits potential as a novel anticancer immunotherapy for CRC, possibly through a synergistic effect when combined with PD-1 blocker treatments. This study may contribute to the ongoing development of anticancer immunotherapies targeting refractory CRC.

scholarly journals Visualization of human T lymphocyte-mediated eradication of cancer cells in vivo

2020 ◽  
Vol 117 (37) ◽  
pp. 22910-22919
Author(s):  
Xingkang He ◽  
Xin Yin ◽  
Jing Wu ◽  
Stina L. Wickström ◽  
Yanhong Duo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Cancer Patients ◽  
Metastatic Cancer ◽  
Car T Cells ◽  
Human T Cell ◽  
Car T ◽  
Metastatic Cancer Cells

Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.

scholarly journals Discovery Strategies to Maximize the Clinical Potential of T-Cell Engaging Antibodies for the Treatment of Solid Tumors

Antibodies ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 65
Author(s):  
Vladimir Voynov ◽  
Paul J. Adam ◽  
Andrew E. Nixon ◽  
Justin M. Scheer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Clinical Data ◽  
Solid Tumor ◽  
Clinical Studies ◽  
Mode Of Action ◽  
Cytotoxic T Cells ◽  
Molecular Structures ◽  
Bispecific Antibodies

T-cell Engaging bispecific antibodies (TcEs) that can re-direct cytotoxic T-cells to kill cancer cells have been validated in clinical studies. To date, the clinical success with these agents has mainly been seen in hematologic tumor indications. However, an increasing number of TcEs are currently being developed to exploit the potent mode-of-action to treat solid tumor indications, which is more challenging in terms of tumor-cell accessibility and the complexity of the tumor microenvironment (TME). Of particular interest is the potential of TcEs as an immunotherapeutic approach for the treatment of non-immunogenic (often referred to as cold) tumors that do not respond to checkpoint inhibitors such as programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) antibodies. This has led to considerable discovery efforts for, firstly, the identification of tumor selective targeting approaches that can safely re-direct cytotoxic T-cells to cancer cells, and, secondly, bispecific antibodies and their derivatives with drug-like properties that promote a potent cytolytic synapse between T-cells and tumor cells, and in the most advanced TcEs, have IgG-like pharmacokinetics for dosing convenience. Based on encouraging pre-clinical data, a growing number of TcEs against a broad range of targets, and using an array of different molecular structures have entered clinical studies for solid tumor indications, and the first clinical data is beginning to emerge. This review outlines the different approaches that have been taken to date in addressing the challenges of exploiting the TcE mode-of-action for a broad range of solid indications, as well as opportunities for future discovery potential.

scholarly journals IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

2013 ◽  
Vol 210 (3) ◽  
pp. 491-502 ◽  
Author(s):  
Shlomo Z. Ben-Sasson ◽  
Alison Hogg ◽  
Jane Hu-Li ◽  
Paul Wingfield ◽  
Xi Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Granzyme B ◽  
Interleukin 1 ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
In Vivo Models ◽  
Ifn Γ

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

624 IFNγ secreted by tebentafusp (IMCgp100)-redirected T cells inhibits expression of melanin synthesis pathway genes in healthy melanocytes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A660-A660
Author(s):  
Mariantonella Vardeu ◽  
David Depoil ◽  
Camille Britton-Rivet ◽  
Jane Houghton ◽  
Jane Harper ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adverse Events ◽  
Cancer Cells ◽  
Cell Lines ◽  
Uveal Melanoma ◽  
Melanin Synthesis ◽  
Activated T Cells ◽  
Melanoma Cancer ◽  
Melanoma Cell Lines

BackgroundTebentafusp (IMCgp100) is a bispecific T cell redirector comprised of an affinity-enhanced TCR recognising melanocyte lineage antigen gp100 and a T cell engaging anti-CD3 scFv domain. Tebentafusp has shown activity as monotherapy in advanced cutaneous and uveal melanoma (Middleton et al., ASCO 2019), and we have previously reported that over half of uveal melanoma patients treated with tebentafusp display melanocyte-related adverse events (MRAE). These include vitiligo/skin hypopigmentation, leukotrichia, and hyperpigmentation and, collectively, are associated with better overall survival in uveal patients receiving tebentafusp (Orloff et al, AACR 2020). In this study, we dissected the mechanisms by which tebentafusp may induce MRAE and highlight the potential clinical significance.MethodsIn vitro studies were conducted to assess the direct and indirect effects of tebentafusp on epidermal melanocytes from healthy donors. Expression of gp100 and the gp100:HLA*02:01 target complex by melanocytes were quantified at the mRNA level and on the cell surface by confocal microscopy, respectively. Melanocytes co-cultured with PBMC and increasing concentrations of tebentafusp were assessed for their susceptibility to lysis and/or ability to stimulate cytokine production. These readouts were compared to gp100-positive and negative melanoma cancer cell lines. Melanin production by melanocytes was quantified and the melanin synthesis pathway interrogated at the mRNA and protein level following exposure to secretomes from tebentafusp-redirected PBMC against melanoma cancer cells.ResultsHealthy melanocytes expressed 2 to 3-fold lower levels of gp100 peptide-HLA complexes on their surface compared to gp100-positive melanoma cell lines. In the presence of tebentafusp, this lower target expression translated into 3–6 fold lower levels of IFNγ and more than 100 fold lower granzyme B production by redirected T cells and these melanocytes were resistant to direct tebentafusp-induced killing (EC50 for melanocytes greater than 1nM vs EC50 melanoma cell lines of 23–50 pM). Supernatants from T cells activated in response to melanoma cancer cells by tebentafusp downregulated the melanin content of healthy melanocytes (20–30% reduction). Western blotting revealed 30–40% inhibition of two key components of the melanin synthesis pathway; the tyrosinase-related protein (TRP)-1 and TRP-2. This inhibition was reversed by blocking IFNγ in supernatants from activated T cells.ConclusionsMRAEs, especially vitiligo, associated with response to tebentafusp, may be explained, at least in part, by the downregulation of melanin biosynthesis pathway genes by IFNγ secreted by tebentafusp-activated T cells.Ethics ApprovalThe study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226ReferencesMiddleton, et al., Relationship between clinical efficacy and AEs of IMCgp100, a novel bispecific TCR–anti-CD3, in patients with advanced melanoma. Journal of Clinical Oncology. 2019.Orloff, et al., Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma. AACR (American Association for Cancer Research), 2020.

Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 477-477
Author(s):  
Erica Dander ◽  
Giuseppina Li Pira ◽  
Ettore Biagi ◽  
Fabrizio Manca ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Target Cells ◽  
Pulsed Dc ◽  
Ifn Γ ◽  
T Cell Lines

Abstract BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines. METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines. RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection. DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 588-588
Author(s):  
Karrune Woan ◽  
Fengdong Cheng ◽  
Hongwei Wang ◽  
Jennifer Rock-Klotz ◽  
Zi Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Egfp Expression ◽  
In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

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