Sensitized CD8+ T Cells Fail To Control Organism Burden but Accelerate the Onset of Lung Injury during Pneumocystis carinii Pneumonia

ABSTRACT While CD8+ cells have been shown to contribute to lung injury during Pneumocystis carinii pneumonia (PCP), there are conflicting reports concerning the ability of CD8+ cells to kill P. carinii. To address these two issues, we studied the effect of the presence of CD8+ cells in two mouse models of PCP. In the reconstituted SCID mouse model, depletion of CD8+ cells in addition to CD4+ cells after reconstitution did not result in increased numbers of P. carinii cysts compared to the numbers of cysts in mice with only CD4+ cells depleted. This result was observed regardless of whether the mice were reconstituted with naïve or P. carinii-sensitized lymphocytes. In contrast, reconstitution with sensitized lymphocytes resulted in more rapid onset of lung injury that was dependent on the presence of CD8+ cells. The course of organism replication over a 6-week period was also examined in the CD4+-T-cell-depleted and CD4+- and CD8+-T-cell-depleted mouse model of PCP. Again, the organism burdens were identical at all times regardless of whether CD8+ cells were present. Thus, in the absence of CD4+ T cells, CD8+ T cells are a key contributor to the inflammatory lung injury associated with PCP. However, we were unable to demonstrate an in vivo effect of these cells on the course of P. carinii infection.

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Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽

10.1182/blood.v106.11.5256.5256 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5256-5256

Author(s):

Doug Cipkala ◽

Kelly McQuown ◽

Lindsay Hendey ◽

Michael Boyer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Mouse Model ◽

Scid Mouse ◽

In Vitro Cytotoxicity ◽

Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

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Transgenic Expression of Inducible Caspase9 Suicide Gene for In Vivo Elimination of Antigen Specific Cytotoxic T Cells (CTLs) Engineered To Produce Cytokines.

Blood ◽

10.1182/blood.v108.11.137.137 ◽

2006 ◽

Vol 108 (11) ◽

pp. 137-137

Author(s):

C. Quintarelli ◽

V. Juan ◽

B. Savoldo ◽

G. Giordano ◽

A. Foster ◽

...

Keyword(s):

T Cells ◽

Mouse Model ◽

Scid Mouse ◽

Cytotoxic T Cells ◽

Safety Margin ◽

Suicide Gene ◽

Cytokine Release ◽

Transgenic Expression ◽

Scid Mouse Model

Abstract Transgenic expression of cytokines such as IL2 and IL15 by tumor specific cytotoxic T cells (CTLs) can improve the survival and expansion of these cells within the tumor microenvironment. Using our model of tumor antigen-Epstein-Barr virus-specific CTLs (EBV-CTLs), we found that transgenic production of either cytokine could sustain CTL expansion ex vivo, and improve their anti-tumor effect in vivo in a SCID mouse model. Although the proliferation of cytokine gene transduced CTLs remained antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous, antigen-independent, growth. We constructed a novel suicide gene based on the inducible caspase9 gene [Straathof et al Blood 2005:105;4247] and showed that EBV-CTLs expressing this suicide gene can be eliminated after exposure to the small molecule chemical inducer of dimerization (CID) AP20187. However, it has been suggested that the anti-apoptotic effects of transgenic cytokine expression would counteract the pro-apoptotic action of icaspase9. To discover the general applicability of our inducible suicide gene approach, we generated tricistronic retroviral vectors encoding full length hIL2 or hIL15, a truncated human CD34 molecule (DCD34) as a marker, and the inducible Caspase9 suicide gene. The three genes were linked using 2A peptide cleavable sequences, which allows equal expression of the 3 transgenes. Established EBV-CTLs were transduced either with IL2.DCD34.iCasp9 or IL15.DCD34.iCasp9 or DCD34 empty vector. After stimulation with the antigen (LCLs) at a E:T ratio of 1:1, we observed significant expansion of CTLs/IL2.DCD34.iCasp9 (221 fold expansion, range 84–452) and IL15.DCD34.iCasp9 (182, range 48–355) after 28 days of culture, while CTLs/DCD34 maintained without cytokines had <2 fold expansion. Cytokine release after antigen stimulation was 6.4±3.4 pg/mL and 157±99pg/mL for IL15 and IL2, respectively. Transgenic CTLs maintained the same phenotype as control T cells (>90% CD3+/CD8+) and remained polyclonal as assessed by staining for the TCRVb repertoire. To evaluate the cytotoxic capacity of the suicide gene, control and transgenic CTLs were incubated with CID AP20187 at 20nM. After 24 hours the survival of transgenic CTLs was estimated from residual CD34+ expression. For both CTLs/IL2.DCD34.iCasp9 and CTLs/IL15.DCD34.iCasp9 the percentage of CD34+ cells was reduced by >1.5 logs. Moreover, the few residual cells were CD34dim, and produced no measurable cytokine release as measured by ELISA. To assess activity in vivo, we used an LCL-engrafted SCID mouse model, injected with CTLs transgenic for the firefly-luciferase gene. These cells were tracked in vivo using the Xenogen-IVIS bioluminescence system. We found that CTLs transgenic for either IL2 or IL15 both migrated to the tumor site, and had increased expansion compared to DCD34 control CTLs. Mice were then treated with 2 −3 doses of CID AP20187 (50 mg/mice i.p). The bioluminescence signal diminished to background levels by 48hr, suggesting efficient elimination of transgenic CTLs. In conclusion, these data indicate that induction of a transgenic caspase9 suicide gene can effectively destroy adoptively transferred T cells, even when these lymphocytes are expressing anti-apoptotic cytokines such as IL2 and IL15. This approach may increase the safety margin of gene modified CTL therapies.

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T cell lessons from the RA synovium SCID mouse model: synovial tissue rich in CD3 T cells lacks response to CTLA4-Ig, but is successfully treated with anti-IL-17 antibodies

Annals of the Rheumatic Diseases ◽

10.1136/ard.2010.149013.15 ◽

2011 ◽

Vol 70 (Suppl 2) ◽

pp. A73-A74

Author(s):

M. I. Koenders ◽

R. J. Marijnissen ◽

S. Abdollahi-Roodsaz ◽

F. E. D. Padova ◽

A. H. Boots ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Synovial Tissue ◽

Scid Mouse ◽

Scid Mouse Model

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Co-Administration Of Gene-Modified CD4+ T Cells Targeting HLA Class I-Restricted WT1 Epitope Diversely Enhances The Antitumor Effect Mediated By Redirected T-Cell Based Adoptive Immunotherapy Against Human Leukemia

Blood ◽

10.1182/blood.v122.21.144.144 ◽

2013 ◽

Vol 122 (21) ◽

pp. 144-144

Author(s):

Hiroshi Fujiwara ◽

Fumihiro Ochi ◽

Toshiki Ochi ◽

Hiroaki Asai ◽

Yukihiro Miyazaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Cd8 T Cells ◽

Adoptive Immunotherapy ◽

Cd4 T Cells ◽

Hla Class I ◽

Class I ◽

Leukemia Cells

Abstract Purpose In the context of redirected T-cell based antitumor adoptive immunotherapy, the therapeutic roles played by co-infused CD4+ T cells genetically redirected to the predefined HLA class I-restricted epitope which had been originally recognized by effector CD8+ T cells has not yet been fully discussed. In this study, using an HLA class I-restricted WT1 -specific T-cell receptor (TCR) gene transfer, we in detail examined antileukemia functionality mediated by these gene-modified CD4+ T cells co-infused with similarly gene-modified effector CD8+ T cells as the redirected T cell-based adoptive immunotherapy. Methods Using our unique retroviral vector expressing HLA-A*2402-restricted and WT1235-243-specific TCR a/b genes and shRNAs for endogenous TCRs (WT1-siTCR vector), we genetically modified both CD4+ and CD8+ T cells from the same healthy donor or leukemia patients (termed WT1-siTCR/CD4 and WT1-siTCR/CD8, respectively). First, target-responsive cellular outputs mediated by WT1-siTCR/CD4 was thoroughly examined using flowcytometry, ELISA, 51Cr-release assay, CFSE dilution assay and bioluminescence assay. Next we similarly assessed impacts of WT1-siTCR/CD4 on the antileukemia functionality mediated by concurrentWT1-siTCR/CD8 both in vitro and in vivo. Eventually, we assessed the in vivo therapeutic efficacy of combined administration of WT1-siTCR/CD8 with WT1-siTCR/CD4 using a xenografted mouse model. Results The transcription factor profile demonstrated that WT1-siTCR/CD4 turned a terminal effector, but not regulatory phenotype. Activated WT1-siTCR/CD4 expressed cell-surface CD40L. Target-responsive cytokine production profile of WT1-siTCR/CD4 represented the Th1 helper function in the context of HLA-A*2402. HLA class II molecules expressed by leukemia cells facilitated the recognition of leukemia cells by WT1-siTCR/CD4 in the context of HLA-A*2402. WT1-siTCR/CD4 displayed the delayed cytocidal activity determined by 51Cr release assay. WT1-siTCR/CD4 could produce IFN-g in response to freshly isolated leukemia cells. WT1-siTCR/CD4 displayed the leukemia trafficking activity in vivo. WT1-siTCR/CD4 represented the potential to migrate into bone marrow via CXCR4/CXCL12 axis both in vitro and in vivo. Concurrent WT1-siTCR/CD4 augmented IFN-g production and cytotoxic degranulation mediated by WT1-siTCR/CD8 in response to the cognate epitope via humoral factors. Consequently, the cytocidal activity against autologous leukemia cells mediated by WT1-siTCR/CD8 was augmented in the presence of WT1-siTCR/CD4, both of them generated from normal lymphocytes of the same patient with leukemia in a complete remission. Upon the target recognition, activated WT1-siTCR/CD4 recruited WT1-siTCR/CD8 via CCL3/4-CCR5 axis. Proliferative response and differentiation into central memory T-cell subset mediated by WT1-siTCR/CD8 in response to the cognate epitope and leukemia cells were enhanced in the presence of autologousWT1-siTCR/CD4, but not gene-modified CD4+ T cells (NGM-CD4). CD127 expression on activated WT1-siTCR/CD8 also increased in parallel to this differentiation. Co-infused WT1-siTCR/CD4 augmented the tumor trafficking and persistence of WT1-siTCR/CD8 in vivo, resulting in the greater suppression of leukemia cells in a xenografted mouse model. Finally, in the therapeutic mouse model, co-infusion of WT1-siTCR/CD8 with of WT1-siTCR/CD4 significantly suppressed the growth of inoculated leukemia cells compared to that in mice received co-infusion of WT1-siTCR/CD8 with NGM-CD4 (Fig.1). Correlation between the therapeutic efficacy and survival of infused gene-modified T cells was also observed. Conclusion In results, the combined infusion of WT1-siTCR/CD8 with WT1-siTCR/CD4, but not NGM-CD4 obviously demonstrates the enhanced antileukemia efficacy via diverse mechanisms. Now we have just started a clinical trial using gene-modified T cells with WT1-siTCR vector for the treatment of patients with refractory acute myeloid leukemia and myeloid dysplastic syndrome. Because redirected T cells employed in this trial encompassed both WT1-siTCR/CD4 and WT1-siTCR/CD8, we are planning to clinically verify the significance of WT1-siTCR/CD4 in the redirected T cell-based antileukemia adoptive immunotherapy. (Fig.1) Disclosures: No relevant conflicts of interest to declare.

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Bromodomain and Extraterminal (BET) Domain Inhibition with PLX51107 and PLX2853 Improves Survival and Decreases Acute GVHD Severity in Murine Models

Blood ◽

10.1182/blood-2019-122455 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4429-4429

Author(s):

Hannah K. Choe ◽

Yandi Gao ◽

Katiri Snyder ◽

Ben Powell ◽

Gideon Bollag ◽

...

Keyword(s):

T Cells ◽

Biological Activity ◽

T Cell ◽

Mouse Model ◽

Mouse Models ◽

Cd8 T Cells ◽

In Vitro Studies ◽

Oral Gavage

Introduction: Acute Graft-versus-Host Disease (aGVHD) affects 30-70% of all allogeneic stem cell transplant (alloSCT) recipients, contributing to high non-relapse mortality. aGVHD is due to donor T cell cytotoxic inflammatory effects stimulated by exposure to foreign host antigens. Epigenetic modulation by BET inhibition boasts anti-inflammatory effects. BET proteins are a class of epigenetic "reader molecules," functioning as key mediators of transcription. BET proteins regulate inflammatory gene expression and are targetable. BET inhibition decreases NF-kB dependent cytokine expression and Th1/Th17 differentiation without affecting regulatory T cell differentiation. BET inhibition disrupts BRD4 interaction with the acetylated component of NF-kB, RelA. Therefore, we hypothesize that BET inhibition is a feasible and effective strategy to mitigate T cell mediated aGVHD inflammation. In this study, we have used PLX51107 and PLX2853 - novel BET inhibitors developed by Plexxikon Inc that possess a unique binding mode and high bioavailability ideal for preclinical evaluation. PLX2853 (second generation) was designed to increase potency and improve tolerability. Materials and Methods: aGVHD allogeneic mouse model: Lethally irradiated (1200 cGy) B6D2F1 recipients received T cell depleted bone marrow cells (TCD-BM, 10x106) and CD45.1+ B6 splenocytes (15x106) intravenously via tail vein injection. Recipients were treated by oral gavage, 3x weekly with PLX51107 (10 mg/kg), PLX2853 (4 mg/kg) or vehicle. We evaluated two dosing schedules - starting at day +1 or day +7 after transplant in the PLX51107 studies and day +1 only after infusion of allogeneic CD45.1+ B6 splenocytes in the PLX2853 studies. Survival and clinical aGVHD scores were assessed. Xenogeneic mouse model: NSG mice were conditioned with 50 cGy X-ray irradiation and injected with 15-20x106 human PBMCs and treated with PLX2853 (4 mg/kg) or vehicle starting day +1. Survival was assessed. Graft-versus-Tumor model: In vivo: Lethally irradiated F1 mice were intravenously injected with firefly luciferase-transduced murine mastocytoma P815 cells on day 0 with B6 TCD-BM ± allogeneic B6 splenocytes (n=8 per cohort). Recipients were treated with PLX2853 (4 mg/kg) or vehicle (oral gavage, 3x weekly) starting at day +1 post-transplant. Survival was assessed. In vitro: Murine CD45.1 B6 CD8+ T cells were stimulated in vitro with PMA/Ionomycin ± PLX2853 (10nM) for 5 days and then degranulation was analyzed in response to P815 tumor challenge to evaluate CTL capacity via flow cytometry of intracellular CD107a expression. Study designs are illustrated in the Figure (Panel A). Results: PLX51107 demonstrates potent biological activity and improves survival in a murine model of aGVHD. Administration of PLX51107 dramatically improved survival of recipient mice (B) and reduced aGVHD clinical scores (C). PLX2853 significantly improves survival in multiple mouse models of aGVHD. Our in-vitro data show that PLX2853 (IC50 ~10nM) is more potent than PLX51107 (IC50 ~500nM). Therefore, we validated the biological activity and efficacy of PLX2853 in mouse models of aGVHD. PLX2853 significantly prolonged survival of allogeneic transplanted recipient mice (D) and resulted in reduced clinical scores (E). Recipient mice in the xenogeneic mouse model showed improved survival with PLX2853 treatment (F). PLX2853 maintains Graft Versus Tumor response in vivo and does not abrogate CD8+ cytotoxic T lymphocyte (CTL) responses in vitro. BET inhibition retained beneficial GVT effects as seen by improvement in survival comparable to vehicle group (G). PLX2853 treated CD8+ T cells showed comparable degranulation to control as measured by CD107a mobilization (H, I). These results suggest that BET inhibition does not abrogate CD8+ CTL capacity, correlating to retention of GVT effects observed in vivo. Conclusions: PLX51107 was well tolerated at both day +1 and +7 initiation, demonstrating the future feasibility of BET inhibition as a prophylactic or therapeutic strategy for aGVHD. PLX2853 given at lower doses demonstrated similar improvements in survival and GVHD scoring, consistent with increased potency. Preliminary in vivo and in vitro studies of PLX2853 on GVT and on CD8+ CTLs show that PLX2853 retains GVT effects. Additional mechanistic in vivo and in vitro studies are ongoing. Figure Disclosures Powell: Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment.

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Prevention of Acute Graft-Versus-Host Disease in Human/Mouse Xenogeneic SCID Mouse Model by Humanized Anti-CD74 Monoclonal Antibody, Milatuzumab

Blood ◽

10.1182/blood.v120.21.4105.4105 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4105-4105

Author(s):

Xiaochuan Chen ◽

Chien-Hsing Chang ◽

Rhona Stein ◽

Thomas M Cardillo ◽

David V. Gold ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Scid Mouse ◽

Acute Gvhd ◽

T Cell Subsets ◽

Graft Versus Host ◽

Cell Immunity ◽

Scid Mouse Model ◽

Ifn Γ

Abstract Abstract 4105 Introduction: Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Prevention and treatment of GVHD remains a major challenge, because current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, leading to severe infections and disease relapse. Thus, novel anti-GVHD agents that can spare protective T-cell memory are critically needed. Milatuzumab (hLL1) is a humanized anti-CD74 antagonist IgG1κ monoclonal antibody (mAb) currently under clinical investigation as a therapeutic mAb for relapsed or refractory B-cell malignancies. Since CD74 is widely expressed in both hematopoietic and non-hematopoietic antigen-presenting cells (APCs), and because host APCs, especially non-hematopoietic APCs, play an important role in initiating GVHD, whereas donor APCs contribute and are required to maximize GVHD, we reasoned milatuzumab could affect recipient or donor APCs, thereby modulating GVHD. We report herein the in vitro effect of milatuzumab on the survival and function of human blood APCs and T cells, including CMV-specific T cells, and the in vivo therapeutic efficacy on preventing acute GVHD in a humanized SCID mouse model. Methods: The effects of milatuzumab on APCs and T cells in human peripheral blood mononuclear cells (PBMCs) were assessed by multi-color flow cytometry. The effect of milatuzumab on the proliferation of T cells and T cell subsets in allogeneic mixed lymphocyte reactions (allo-MLRs) was measured by CFSE labeling of allogeneic PBMCs that were mixed to each other and cultured for 11 days before flow cytometric analysis of the proliferating cells that lost fluorescence. The impact of milatuzumab on preexisting anti-viral T-cell immunity was evaluated by intracellular staining of CD3+CD8+IFN-γ+T cells in allo-MLRs upon stimulation with HLA-A2-restricted cytomegalovirus (CMV) pp65 peptide (NLVPMVATV). The therapeutic effect of milatuzumab on acute GVHD was evaluated in a human PBMC-transplanted SCID mouse model, in which GVHD is developed and mediated by engrafted human T cells and DCs. Results: Milatuzumab moderately reduces the number of myeloid DC type 1 (mDC1), myeloid DC type 2 (mDC2), and B cells in PBMCs, but has little effect on plasmacytoid DC (pDC), monocytes, or T cells, which correlates with the level of CD74 expression on these cells. As a consequence, milatuzumab inhibits the proliferation of total T cells, CD4 and CD8 T cell subsets in allo-MLRs. In a human/mouse xenogeneic SCID mouse model, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses the serum levels of human interferon-g and IL-5, eliminates the infiltration of human lymphocytes in GVHD target organs (lung, liver and spleen), and significantly promotes the survival of animals (90% versus 20% for controls, P=0.0012). Furthermore, exposure to milatuzumab does not affect the number of CMV-specific, IFN-γ-producing, human CD8+ T cells in allo-MLRs. Conclusion: CD74 is a potential target for antibody-mediated mitigation of GVHD, as demonstrated by the encouraging results obtained with milatuzumab. Further exploration of milatuzumab as a new therapeutic agent for GVHD is warranted. Disclosures: Cardillo: Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

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The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases the Severity of Acute Lung Injury After Allogeneic Hematopoietic Cell Transplantation.

Blood ◽

10.1182/blood.v120.21.3006.3006 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3006-3006

Author(s):

Sabarinath Venniyil Radhakrishnan ◽

Senthilnathan Palaniyandi ◽

Fridrik Karlsson ◽

Elisabeth Huber ◽

Gerhard Hildebrandt

Keyword(s):

T Cells ◽

T Cell ◽

Lung Injury ◽

Cd8 T Cells ◽

Hematopoietic Cell Transplantation ◽

Lung Pathology ◽

Allogeneic Hct ◽

Cell Responses

Abstract Abstract 3006 Background: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges, with acute lung injury being associated with significant morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that the PHI DMOG (Dimethyl oxalyl glycine) may reduce lung injury evolving after murine allogeneic HCT. Methods: 14 week old BALB/c were conditioned with 750 cGy single dose TBI, followed by transplantation of 5×10E6 bone marrow cells and 4×10E6 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. DMOG in PBS was given to both syngeneic and allogeneic recipients at 8mg per mice ip on alternate days for the first two weeks post-transplant and then twice a week till day 50, while syngeneic and allogeneic controls received PBS only. Animals were monitored for clinical GVHD and survival. At day 50 post HCT, gut, liver and lung pathology as well as BAL fluid T cell numbers were assessed by using a scoring system quantifying severity and extent of parenchymal and peribronchial/perivascular infiltrates and flow cytometry, respectively. In addition, T cell expansion was analyzed both in vivo in the spleen on day +7 and in vitro by mixed lymphocyte reaction (MLR). Results: Allogeneic HCT resulted in severe lung pathology in PBS treated animals (mean 118.8±47.19), but was significantly reduced when DMOG was given (36.0±17.99; p < 0.05), and absent in both PBS and DMOG treated syngeneic controls. Consistently, BAL CD4+ and CD8+ T cells in DMOG recipients were decreased by 53.7% and 68.4%, respectively, when compared to PBS controls. Decreased pulmonary injury was associated with a trend towards better overall survival by day 50 (allogeneic DMOG: 59.2% vs allogeneic PBS 40.7%) and all syngeneic recipients survived. GVHD related injury to liver and GI tract at time of analysis was not different between allogeneic groups. Alloreactive proliferative T cell responses in vitro were suppressed in the presence of DMOG at a concentration of 1mM (cpm after 96h: mean 29316±875.9 vs 41735±3085; p < 0.01). In vivo, splenic T cell expansion of CD4+ but not CD8+ T cells was reduced at day +7 as well (p < 0.05). Interestingly, when compared to PBS treated controls reduced mortality of DMOG treated allogeneic HCT recipients could already be seen soon after HCT (day +10: 26.0% vs 11.1%) in association with decreased CD4+ T cell proliferation, while lung injury in this model this early is rather marginal. Conclusion: DMOG treatment decreases the severity of acute noninfectious lung injury and improves overall survival after allogeneic HCT. While impaired early T cell responses likely contribute to improvement of both endpoints, altered recruitment of CD8+ T cells to the lung seems to uniquely affect the development of pulmonary but not gastrointestinal and hepatic injury at later time points. Current studies are ongoing to delineate the effects of DMOG on T cell proliferation and recruitment to different target organs, and to define the role of this novel approach in the prevention and treatment of HCT related complications. Disclosures: No relevant conflicts of interest to declare.

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CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

Journal of Experimental Medicine ◽

10.1084/jem.20021429 ◽

2002 ◽

Vol 197 (1) ◽

pp. 19-26 ◽

Cited By ~ 27

Author(s):

Melanie S. Vacchio ◽

Richard J. Hodes

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tolerance Induction ◽

Peripheral Tolerance ◽

Cd8 Cells ◽

Costimulatory Signal ◽

Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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