Prevention of Rat Cardiac Allograft Rejection by Host Th2 Cell Adoptive Transfer

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 365-365
Author(s):  
Shoba Amarnath ◽  
Hao Chen ◽  
Carliann M Costanzo ◽  
Joanna M Swerczek ◽  
Michael A Solomon ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Cd8 T Cells ◽  
Allograft Rejection ◽  
Ex Vivo ◽  
Cardiac Allograft ◽  
Th2 Cells ◽  
Cell Transfer ◽  
Th2 Cell ◽  
Ifn Γ

Abstract We have recently shown that ex vivo manufactured, rapamycin-resistant donor Th2 cells (Th2.R cells) prevent the rejection of allogeneic bone marrow grafts through a process that involves IL-4-mediated polarization of host T cells towards a Th2 phenotype. Because donor T cell therapy lacks feasibility in the setting of cardiac allograft transplantation using cadaveric donors, we developed an approach to prevent rejection that incorporates host Th2 cell therapy prior to organ transplantation. The aims of the study were to: (1) develop a method for the ex vivo manufacture of rat Th2.R cells and inject such syngeneic Th2.R cells just prior to class I and class II disparate cardiac allografting; (2) determine whether such adoptive host Th2.R cell transfer polarizes post-transplant immunity towards a Th2 phenotype; and (3) evaluate in a preliminary manner whether Th2.R cell transfer reduces graft rejection in a model that uses sub-optimal cyclosporine therapy. Recipient-type (Black Norway, BN) CD4+ T cells were co-stimulated with anti-rat anti-CD3 and –CD28 antibodies in the presence of rapamycin, rrIL4, rhuIL-2, and rhIL7 (3-day culture interval). The Th2.R cell cytokine phenotype was determined by intra-cellular flow cytometry and multi-analyte testing of supernatants obtained after repeat co-stimulation. Th2.R cells were injected (i.v.; 1 × 107 cells) 2–3 hours prior to cardiac allografting (donor strain, Dark Agouti; DA). Cardiac function was evaluated clinically by cardiac palpation through day 28 post-grafting. At day 28, histology studies and immune function studies were performed, including assessment of host-anti-donor alloreactivity. Engraftment controls (cohort #1) and rejection controls (cohort #2) received cyclosporine (CSA) through the full 28 days or only through day 18, respectively; the experimental cohort (#3) received host Th2.R cells followed by the short-course of CSA. Supernatants from the Th2.R cell product contained 1500 pg/ml IL-4 and only 50 pg/ml of IFN-γ; the frequency of IL-4+ cells and IFN-γ+ cells was 10% and 1%, respectively. Clinical rejection was observed in cohort #2 (3/3 subjects) whereas all subjects in cohorts #1 and #3 had full cardiac function through day 28 (cohort #1- 2/3 subjects; cohort #3- 3/3 subjects). Relative to rejection controls, Th2.R cell recipients had a reduced frequency of intra-cardiac CD8+ T cells (%CD8+ T cells, 9.2 ± 6.2 vs. 1.1 ± 0.3; p<0.001) and spleen CD8+ T cells (1.9 ± 0.2 vs. 0.5 ± 0.2; p=0.014). Furthermore, after ex vivo re-stimulation with donor-type dendritic cells, intra-cardiac CD8+ T cells from Th2.R cell recipients tended to produce less IFN-γ relative to rejection controls (pg/ml in supernatant; 15.7 ± 1.4 vs. 44.2 ± 1.4; p=0.07) and tended to produce increased IL-4 relative to rejection controls (1974 ± 140 vs. 260 ± 291; p=0.07). This study demonstrates a feasible and successful methodology for inducing recipient based immunotolerance by generating rapamycin-resistant Th2 cells in the rat species.. Adoptive transfer of host-type Th2.R cells just prior to fully genetically-disparate cardiac allograft transplantation results in a shift towards Th2 cytokines post-transplant, a reduction in the frequency of intra-cardiac T cell infiltration, and shows promise as a novel method to prevent cardiac allograft rejection.

Ex-Vivo Characterization of Polyclonal Memory CD8+ T-Cell Responses to PRAME-Specific Peptides in Patients with Acute Leukemia and Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2910-2910
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Abdul Tawab ◽  
Behnam Jafarpour ◽  
Rhoda Eniafe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
High Avidity ◽  
Cell Responses ◽  
Ifn Γ

Abstract PRAME (Preferentially expressed antigen of melanoma) is aberrantly expressed in hematological malignancies and may be a useful target for immunotherapy in leukemia. We studied CD8+ T-cell responses to four HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and healthy donors, using PRA300/HLA-A*0201 tetramer staining, intracellular cytokine (IC) assay and ex-vivo and cultured ELISPOT analysis. CD8+ T-cells recognizing PRAME peptides were detected directly ex-vivo in 4/10 ALL, 6/10 AML, 3/10 CML patients and 3/10 donors. The frequency of PRAME-specific CD8+ T-cells was greater in patients with AML, CML and ALL than in healthy controls. All peptides were immunogenic in patients, whilst PRA300 was the only immunogenic peptide in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to two or more PRAME epitopes (4/7 vs. 0/23 in individuals with low PRAME expression, P = 0.001), suggesting a PRAME-driven T-cell response. In 2 patients studied PRA300/HLA-A*0201+ CD8+T-cells were found to be a mixture of effector and central memory phenotypes. To determine the functional avidity of the PRAME T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of peptide was measured by IC-IFN-γ staining. High-avidity CD8+ T-cells were defined as those capable of producing IFN-γ in response to the lower concentration of peptide (0.1μM), while low-avidity CD8+ T-cells were those that only produced IFN-γ in response to the higher concentration of peptide (10 μM). Both high and low-avidity CD8+ T-cell responses could be detected for all peptides tested (median 1.05, 0.90, 0.52, 0.40 high/lowavidity ratios for PRA100, PRA142, PRA300 and PRA425 respectively). In patients with high PRAME expression (>0.001 PRAME/ABL) low-avidity CD8+ T-cell responses to PRAME peptides were more prominent than high-avidity responses, suggesting selective deletion of high-avidity T-cells. In contrast, in some patients with levels <0.001 PRAME/ABL, we could detect the presence of high-avidity CD8+ T-cell responses to PRAME. PRAME-specific CD8+ T-cells were further characterized by IC staining for IL-2, IL-4 and IL-10 production and CD107a mobilization (as a marker of cytotoxicity). Following stimulation with the relevant PRAME peptide, there was no significant production of IL-2, IL-4 or IL-10, suggesting a Tc1 effector response but no significant CD107a mobilization was detected despite significant CD107a mobilization in the same patient in response to CMVpp65495. This finding suggests that patients with leukemia have a selective functional impairment of PRAME-specific CD8+ T-cells, consistent with PRAME-specific T cell exhaustion. However, PRAME-specific T-cells were readily expanded in the presence of cytokines in short-term cultures in-vitro to produce IFN-γ, suggesting that it may be possible to improve the functional capacity of PRAME-specific T-cells for therapeutic purposes. These results provide evidence for spontaneous T-cell reactivity against multiple epitopes of PRAME in ALL, AML and CML and support the usefulness of PRAME as a target for immunotherapy in leukemia. The predominance of low-avidity PRAME-specific CD8+ T-cells suggests that achievement of a state of minimal residual disease may be required prior to peptide vaccination to augment T-cell immune surveillance.

Adoptive Transfer of Treg-Depleted Donor Th1 and Th2 Cells Safely Accelerates Alloengraftment After Low-Intensity Chemotherapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 521-521 ◽  
Author(s):  
Daniel H. Fowler ◽  
Miriam E. Mossoba ◽  
Bazetta Blacklock Schuver ◽  
Paula Layton ◽  
Frances T Hakim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Cell Therapy ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
Th2 Cells ◽  
Low Intensity ◽  
Ex Vivo Culture

Abstract Abstract 521 Ex-vivo culture of murine donor CD4+ T cells using rapamycin, co-stimulation, and IL-4 yielded a defined T cell population (T-rapa cells) that favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate these findings, we conducted a multi-center clinical trial (NCT0074490) to evaluate T-rapa cell therapy after allogeneic HCT. T-rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation in media containing IL-4, IL-2, and rapamycin. T-rapa cells had a mixed Th2/Th1 phenotype with minimal Treg content (intra-cellular flow, n=48 products; median transcription factor expression: 11.5% [GATA-3], 5.1% [T-bet], and 0.1% [FoxP3]). Median T-rapa cell cytokine secretion (pg/ml; re-stimulation at harvest) was 1.3 [IL-4], 20.6 [IL-5], 9.7 [IL-10], 23.7 [IL-13], 34.7 [IFN-g], and 17.1 [IL-2]. Patients received an HLA-matched sibling, T cell-replete, G-CSF mobilized allograft, and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT). Two protocol arms evaluated T-rapa cell therapy after induction chemotherapy and outpatient, low-intensity preparative chemotherapy (Table I). First, patients (n=25) were accrued to arm A to evaluate T-rapa infusion at d +14 post-HCT; subsequently, accrual was initiated to arm B (n=25) to evaluate T-rapa infusion on d0 of HCT. Arm A was then expanded to n=40 patients. Patients accrued to arms A and B were similar for recipient age, high-risk malignancy diagnosis, chemotherapy refractoriness, and prior regimen number (Table I). Most recipients were not in remission at the time of HCT. High-risk NHL was the most frequent diagnosis (25/65 patients), followed by non-high-risk NHL (11/65), AML/MDS (8/65), myeloma (7/65), CLL (6/65), Hodgkin's disease (5/65), and CML (3/65). Arm A and B recipients had similar mean donor myeloid cell chimerism at d +14, +28, and +100 (arm A, 43%, 74%, and 89%; arm B, 50%, 62%, and 84%). At d +14, arm A and B recipients also had mixed donor T cell chimerism (mean values, 60% in each arm; Table I). At d +28 and +100, T cell chimerism increased in arm A to 80% and 89%; in arm B, these values increased to only 67% and 69%. Four recipients on arm B had < 10% donor T cell chimerism at d +100; in contrast, the lowest donor T cell chimerism value observed at d +100 on arm A was 36%. T-rapa therapy on arm A was relatively safe as there was: no engraftment syndrome, a 10% rate of acute grade II to IV GVHD, a 67% incidence of chronic GVHD, and no transplant-related mortality (Table I). On arm A, 37.5% (15/40) of recipients are in sustained complete remission, with a median survival probability of 63.6% at 24 months post-HCT. Therefore, pre-emptive donor lymphocyte infusion with ex-vivo manufactured T-rapa cells that express a balanced Th2/Th1 effector phenotype represents a novel approach to safely accelerate alloengraftment and harness allogeneic GVT effects after low-intensity conditioning.Table IArm AArm BLow-Intensity Regimen    Induction Chemotherapy1EPOCH-FREPOCH-FR    2Terminal Chemotherapy3Flu (120 mg/m2)EPOCH-FRCy (1200 mg/m2)T-Rapa Cell TimingD +14 post-HCTD 0 of HCTPatient Characteristics    & of Patients Accrued4025    Age (median, range)55 (25–67)51 (32–66)    & of Prior Regimens3 (1–6)3 (1–8)    High-Risk Malignancy65% (26/40)52% (13/25)    Chemotherapy Refractory50% (20/40)48% (12/25)    CR (at time of HCT)25% (10/40)8% (2/25)% Donor T Cell ChimerismMean Median (Range)Mean Median (Range)Day 14 post-HCT6061(8–97)6060(4–100)Day 28 post-HCT8089(27–100)6773(10–100)Day 100 post-HCT8993(36–100)6982(0–100)Clinical Results    Engraftment Syndrome0% (0/40)0% (0/25)    Acute GVHD10% (4/40)23% (5/22)    Chronic GVHD67% (22/33)75% (15/20)    Complete Remission38% (15/40)28% (7/25)    Transplant-related Mortality0% (0/40)0% (0/25)    Percent Survival65% (26/40)40% (10/25)    Median Survival27.5 mo11.2 mo    Survival Prob. at 24 mo63.6%44.0%1EPOCH-FR, EPOCH with fludarabine (Flu) and rituximab.2Terminal (preparative) chemotherapy administered one week prior to HCT.3Flu/Cy [cyclophosphamide] doses are total doses, given over 4 days (Cy dose is 75% lower than 4800 mg/m2 “reduced-intensity” Cy dose). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modulation of LIGHT-HVEM Costimulation Prolongs Cardiac Allograft Survival

2002 ◽  
Vol 195 (6) ◽  
pp. 795-800 ◽  
Author(s):  
Qunrui Ye ◽  
Christopher C. Fraser ◽  
Wei Gao ◽  
Liqing Wang ◽  
Samantha J. Busfield ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Allograft Rejection ◽  
Cell Activation ◽  
Cardiac Allograft ◽  
Allograft Survival ◽  
Activated T Cells ◽  
Cardiac Allograft Rejection ◽  
Ifn Γ

LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT−/− mice by homologous recombination. The mean survival of fully major histocompatibility complex–mismatched vascularized cardiac allografts in LIGHT−/− mice (10 days, P &lt; 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P &lt; 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT−/− allograft recipients (30 days) was considerably enhanced (P &lt; 0.001) compared with the 10 days of mean survival in either untreated LIGHT−/− mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-γ, plus IFN-γ–induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P &lt; 0.001). Our data suggest that T cell to T cell–mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.

Preclinical efficacy of dendritic cells derived from bone marrow hematopoietic stem cells for the treatment of syngeneic prostate cancer immune cell therapy.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 361-361
Author(s):  
Yunlim Kim ◽  
Choung-Soo Kim ◽  
Bongmin Kim
Keyword(s):  
Prostate Cancer ◽  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
Cell Therapy ◽  
Cd8 T Cells ◽  
Treated Group ◽  
Hematopoietic Stem ◽  
Anti Cancer ◽  
Ifn Γ

361 Background: PCas are characterized by a primary androgen dependent stage, during which growth is promoted by androgen and suppressed by androgen deprivation or blockade, with even metastatic disease being promptly susceptible to hormonal manipulation; however, despite initial success, androgen deprivation therapy eventually fails because of the development of CRPC. Despite the approval of new clinical trials such as docetaxel, enzalutamide and sipuleucel-T, which is a cell therapy drug, the clinical efficacy is limited. Therefore, there is a desperate need for the limited effectiveness of treatment in an aging society and the development of successful treatments for patients who fail treatment. In this study, we investigated the anticancer effect of prostate cancer using anti-cancer immunity cell vaccine based on the next generation dendritic cell immunotherapy. Methods: FACS and ELISA were performed with stem-DC to analyze phenotype and cytokine expression. To quantify the effect on the tumor growth, we constructed a TRAMP-C1 model and observed for safety, efficacy and induction of antigen-specific immune responses. And we also analyzed proliferation and activation of allogeneic-T cells by therapeutic dendritic cells. Results: From the stem-DC, immune-stimulatory cytokine IL-12 and interferone-gamma was measured higher level than monocyte derived DC (Mo-DC). A DC marker CD11c+CD8a+ cell was higher expression in stem-DC than Mo-DC. After DC injection, tumor size was reduced in stem-DC compared to Mono-DC and vehicle group in TRAMP model. To confirm stem-DC/lysate induce more systemic anti-tumor immunity than Mo-DC/lysate, we checked effector T cells in splenic lymphocytes from prostate cancer bearing mice. The frequency of IFN-γ secreting CD8+ T cells in stem-DC treated group was significantly higher than that in Mo-DC treated group. It shows that the therapeutic responses were associated with induction of IFN-γ secreting CD8+ T cells. Conclusions: Stem-DC, which was developed from hematopoietic stem cells in an optimized manner, proved to be effective and stable as an excellent anti-cancer immunotherapeutic agent for prostate cancer.

Graft-Versus-Host Disease or Infection: Rapid Detection of HLA Mismatch-Reactive T Cells Ex Vivo Can Facilitate Diagnosis and Guide Therapy after Allogeneic Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4993-4993
Author(s):  
Eva Distler ◽  
Simone Thomas ◽  
Elke Schuerer ◽  
Cedrik Britten ◽  
Martin Schuler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Rapid Detection ◽  
Clinical Course ◽  
Ex Vivo ◽  
Hla Alleles ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract Diagnosis of graft-versus-host disease (GVHD) is mainly based on clinical features and on tissue biopsies. However, clinicians and pathologists are well aware of cases, in which GVHD cannot be distinguished from infections arising from severe immunodeficiency after allogeneic stem-cell transplantation (SCT). This may pose a deep therapeutic dilemma of whether to modify immunosuppressive treatment or to use donor lymphocyte infusion (DLI) for promoting anti-microbial immunity. We observed a 68-year-old patient with myelodysplastic syndrome who developed acute GVHD grade II of skin and gut at d+16 after T-cell depleted reduced-intensity SCT (Fig. 1). GVHD was confirmed by histology and responded to prednisolone therapy. From d+90 to d+240, the patient suffered from massive diarrhea (>2L per day) and recurrent episodes of lower gastrointestinal bleeding. Histopathology analysis on gut biopsies showed a heterogeneous picture with signs of GVHD and ulcerative inflammation. In stool screening, we isolated norovirus type 2 (ELISA, PCR) between d+111 and d+229, thereby confirming the longest infection with this virus ever reported. Tapering immunosuppression did not improve diarrhea, and the patient required intensive care due to serious fluid imbalance. Because of severe lymphopenia (<100 per μL CD3 T cells), we considered DLI therapy to promote T cell reconstitution and norovirus clearance. To balance the risk for DLI-induced exacerbation of GVHD, we first analyzed peripheral blood CD8 T cells for anti-recipient reactivity ex vivo by IFN-γ ELISPOT assay. Due to the limited availability of recipient cells and a single HLA disparity between donor (B*3508) and patient (B*3503), we used HLA-deficient K562 cells transfected with mismatched (B*3503) or matched (A*0201) HLA alleles as antigen-presenting cells. Post-transplant CD8 T cells specifically recognized disparate HLA-B*3503 (Fig. 1), but not shared HLA-A*0201. Mismatch-reactive CD8 T cells were detectable at significant numbers (71/105) during the first episode of GVHD and correlated closely with the intensity of diarrhea beyond d+110. Maximum anti-HLA-B*3503 reactivity (439/105) on d+205 was in the range of IFN-γ spot production obtained in mitogen-stimulated controls. Considering this vigorous alloreactivity, we were concerned that scheduled DLI would boost GVHD rather than facilitating norovirus clearance. Therefore, we decided to omit DLI. Patient’s clinical condition improved spontaneously around d+240, and diarrhea did not recur thereafter. In conclusion, we have established a new T cell assay for the rapid detection of anti-HLA mismatch reactivity using K562-HLA transfectants as substitutes for patient cells. In the meantime, we have validated this assay in two further HLA-incompatible donor-patient pairs and generated off-the-shelf K562 transfectants for more than 15 HLA alleles. Ex vivo alloreactivity toward mismatch HLA might be a surrogate marker to facilitate GVHD diagnosis and guide therapy in ambiguous clinical situations, such as the coincident viral infection described herein. Fig. 1: Clinical course and monitoring of all-HLA-B*3503 reactive CD8 T cells ex vivo Fig. 1:. Clinical course and monitoring of all-HLA-B*3503 reactive CD8 T cells ex vivo

The Induction of Inhibitory Pathways in Dendritic Cells May Hamper the Efficient Activation of Anti-Leukemia T Cells within Chemotherapy-Induced Immunogenic Cell Death

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1019-1019
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Elisa Orioli ◽  
Elena De Marchi ◽  
Sabina Sangaletti ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Atp Release ◽  
Inhibitory Pathways ◽  
Ifn Γ

Abstract BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis. METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release. RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype. CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

scholarly journals Rapid Effector Function in CD8+ Memory T Cells

1997 ◽  
Vol 186 (6) ◽  
pp. 859-865 ◽  
Author(s):  
Ajit Lalvani ◽  
Roger Brookes ◽  
Sophie Hambleton ◽  
Warwick J. Britton ◽  
Adrian V.S. Hill ◽  
...  
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Influenza Virus Infection ◽  
Immunological Memory ◽  
Effector Function ◽  
Memory State ◽  
Memory Cd8 T Cells ◽  
Ifn Γ

The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon γ (IFN-γ) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-γ release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I–restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.

scholarly journals HIV-Specific Cd8+ T Cells Produce Antiviral Cytokines but Are Impaired in Cytolytic Function

2000 ◽  
Vol 192 (1) ◽  
pp. 63-76 ◽  
Author(s):  
Victor Appay ◽  
Douglas F. Nixon ◽  
Sean M. Donahoe ◽  
Geraldine M.A. Gillespie ◽  
Tao Dong ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Viral Infections ◽  
Ex Vivo ◽  
Hla Class I ◽  
Cytolytic Activity ◽  
Specific Lysis ◽  
Exact Function ◽  
Tnf Α ◽  
Ifn Γ

The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8+ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8+ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8+ T cells compared with cytomegalovirus (CMV)-specific CD8+ T cells in HIV chronic infection. We show that the majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8+ T cells express significantly lower levels of perforin than CMV-specific CD8+ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8+ T cells are impaired in cytolytic activity.

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