Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2176-2176
Author(s):  
Attilio Bondanza ◽  
Lothar Hambach ◽  
Shin Kaneko ◽  
Sara Mastaglio ◽  
Bart Nijmeijer ◽  
...  
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Suicide Gene ◽  
Effector T Cells ◽  
Donor T Cells ◽  
Specific Effector ◽  
Ifn Γ ◽  
Homeostatic Cytokines

Abstract In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.

Efficient Induction and Isolation of CMV-Specific CD8+ T Cells from CMV Seronegative Donors for the Treatment of CMV Reactivation in CMV Seropositive Patients Transplanted with a CMV Seronegative Donor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1053-1053 ◽  
Author(s):  
Inge Jedema ◽  
Marian van de Meent ◽  
Pim L.J. van der Heiden ◽  
Erik W.A. Marijt ◽  
Pauline Meij ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Memory T Cells ◽  
Immunomagnetic Bead ◽  
Healthy Donors ◽  
Donor T Cells ◽  
The Impact ◽  
Cmv Reactivation

Abstract Cytomegalovirus (CMV) disease is a significant cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT) in CMV seropositive (CMV+) patients. In a recent cohort of CMV+ patients, we investigated the impact of donor CMV serostatus on the severity of CMV reactivation after T cell depleted allo-SCT. A high incidence of CMV related mortality was seen in patients transplanted with a CMV- donor (5/20) whereas no CMV-related deaths occurred after transplantation with a CMV+ donor (0/20). In most CMV+ patients transplanted with a CMV+ donor reconstitution with CMV specific (memory) T cells was found. We recently performed a phase I/II clinical study using isolated CMV-specific CD8+ memory donor T cells for the treatment of patients with persistent CMV reactivation despite seropositivity of the donor. In this study we demonstrated the feasibility of isolating and selecting CMV-specific CD8+ memory T cells from CMV+ donors using the interferon-gamma (IFNg) capture assay and CliniMACS isolation after peptide stimulation of the CMV-specific donor T cells. We have illustrated the in-vivo potential of these T cells after adoptive transfer in 5 patients, resulting in clearance of the CMV load. However, no suitable method was available for the induction of primary immune responses against CMV for the treatment of persistent CMV reactivation in patients transplanted with a CMV- donor. In the current study we investigated the possibility to induce and isolate CMV-specific T cells from CMV- healthy donors by in-vitro priming and selection. We used as responder cells CD14- CD45RO- PBMC from HLA-A1, A2, A3, B7, or B8 positive CMV- healthy donors (n=10). By CD45RO depletion we removed the majority of regulatory T cells (Tregs) capable of inhibiting the initiation of the response. Mature monocyte-derived dendritic cells (DCs) were loaded with a cocktail containing 1μg of each relevant CMV pp65, pp50, or IE1 derived 9-mer peptide, depending on the HLA type of the donor. Naïve donor T cells were cocultured in a 10/1 ratio with peptide loaded DCs. From day 4 on 5 ng/mL IL-7 and IL-15 was added to the culture. At day 10, the responses were specifically restimulated with peptide loaded autologous PBMC. Cytokines were refreshed twice weekly. At day 20 CMV-specific CD8+ T cells were detected and isolated by specific tetramer staining and flowcytometric cell sorting, by specific pentamer staining and immunomagnetic bead isolation, or further enriched by another restimulation, followed by isolation of CD137 expressing T cells at day 21. In 10/10 CMV seronegative donors CMV specific T cells could be detected at day 20 of the immune response in frequencies ranging from 0.01–0.4%. These tetramer positive cells could be isolated and expanded both in bulk cultures and clonally. Functional CMV-specific T cells against all 3 major immunogenic CMV proteins pp65, pp50, and IE1 were detected and isolated with different dominant responses detected in different donors. In conclusion, we have developed a method for the in-vitro induction and isolation of functional CMV-specific CD8+ T cells from CMV- donors. This may allow the treatment of serious CMV-related complications in CMV+ patients transplanted with a CMV- donor.

Identification of Previously Uncharacterized Delta 4-Positive Inflammatory Plasmacytoid Dendritic Cells That Play Important Roles in Eliciting Graft-Versus-Host Disease in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 337-337 ◽  
Author(s):  
Kazuhiro Mochizuki ◽  
Fang Xie ◽  
Shan He ◽  
Qing Tong ◽  
Yongnian Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Effector T Cells ◽  
Activated T Cells ◽  
Graft Versus Host ◽  
Notch Receptors ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Antigen Presenting

Abstract Abstract 337 Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Host antigen-presenting cells (APCs) are known to be essential for presenting alloantigens to activate donor T cells to become effector cells mediating GVHD after allo-HSCT. However, APCs are heterogeneous populations. The identity of APC subset(s) that directs effector differentiation of alloantigen-activated T cells and by which mechanism this effect may be achieved remain largely unknown. The Notch signaling pathway controls cell proliferation, differentiation and survival. Upon interaction with Notch ligands of the δ-like family (Dll1, Dll3 and Dll4) and Jagged family (J1, J2), Notch receptors (Notch 1, 2, 3, and 4) are cleaved by γ-secretase and translocate into the nucleus to modify gene transcription. We have recently demonstrated that activation of Notch receptors in donor T cells is critical to the production of alloreactive effector T cells producing multiple inflammatory cytokines (e.g., IFN-γ, TNF-α and IL-17) during GVH reaction (Blood 2011). Building on these findings, we hypothesized that: 1) Notch ligand(s) derived from APCs may be important for directing effector differentiation of alloantigen-activated T cells, and 2) the expression of Notch ligand(s) may differentiate the capability of APCs to prime GVH responses. Using mouse models of GVHD, here we report the identification of previously uncharacterized Dll4-positive (Dll4+) inflammatory plasmacytoid dendritic cells (i-pDCs) and their roles in eliciting allogeneic T-cell responses. Host-derived Dll4+ i-pDCs occurred in the spleen of allo-HSCT recipients one day after transplantation, peaked by three days and declined by seven days. In contrast, host-derived inflammatory conventional DCs (i-cDCs) were Dll4-negative (Dll4−) and rapidly diminished by three days after transplantation. Notably, donor-derived DCs which occurred seven days after HSCT did not express Dll4. In vitro mixed lymphocyte-reaction (MLR) assay showed that these host-derived Dll4+ i-pDCs induced approximately 2.5-fold and 7-fold more IFN-γ- and IL-17-producing effector T cells than Dll4− i-cDCs, respectively. Addition of neutralizing antibody specific to Dll4 to the MLR cultures markedly reduced the production of IFN-γ and IL-17 in donor T cells stimulated by host Dll4+ i-pDCs, but had minimal impact on donor T cells cultured in the presence of Dll4− i-cDCs. These results suggest that Dll4+ i-pDCs may play important roles in directing effector differentiation of alloantigen-activated T cells. Further characterization of biological properties of Dll4+ i-pDCs revealed that as compared to unstimulated host pDCs at steady state conditions, Dll4+ i-pDCs expressed higher levels of antigen-presenting and costimulatory molecules, upregulated other Notch ligands (e.g.,J1 and J2) on their surface and produced more Ifnb and Il23. Notably, Dll4+ i-pDCs were mainly located in the spleen and intestine of mice receiving allogeneic HSCT. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cell producing IFN-γ, IL-17 and TNF-α in GVHD target organs (in particular of the intestine), leading to reduction of GVHD and significantly improved survival of mice after allogeneic HSCT. Furthermore, adoptive transfer of in vitro generated Dll4+ i-pDCs caused severe GVHD in MHC-II-deficient mice (in which host DCs are incapable to elicit GVHD). Our findings identify that Dll4+ i-pDCs may represent a previously uncharacterized inflammatory APC population developed during GVH reaction. These Dll4+ i-pDCs and their-derived Dll4 are critical for directing differentiation of alloreactive effector T cells and may be beneficial therapeutic targets for modulating GVHD. Disclosures: No relevant conflicts of interest to declare.

Tracking Genetically Engineered Lymphocytes Long-Term Reveals the Dynamics of T-Cell Immunological Memory

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 263-263
Author(s):  
Giacomo Oliveira ◽  
Eliana Ruggiero ◽  
Maria Teresa Lupo Stanghellini ◽  
Nicoletta Cieri ◽  
Mattia D'Agostino ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Suicide Gene ◽  
T Cell Subsets ◽  
Memory T Cell ◽  
Donor T Cells

Abstract BACKGROUND: Long-term T-cell survival is pivotal for the development of effective therapeutic approaches against pathogens and cancer, since the success of immunotherapy requires the generation of a robust, safe but also durable immune response. Even if it is established that memory cells can survive and persist for years, little is known about the requirements for their long-term persistence. Suicide gene therapy after T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) provides a unique model to study memory T cells. In this setting, patients receive the post-transplant infusion of donor-derived gene-modified memory T lymphocytes retrovirally transduced to express the Herpes Simples Virus Thymidine Kinase (TK) suicide gene and the DLNGFR selection marker. The presence of a safety switch allows the infusion into patients of a broad T-cell repertoire in the absence of immune suppression, while the surface marker enables unambiguous detection and close monitoring of gene-modified cells circulating in treated patients. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the fate of persisting TK cells to shed light on memory T cell dynamics in vivo and to unravel the requirements for long-term persistence directly in humans. RESULTS: We studied 10 adult patients who underwent haplo-HSCT and infusion of suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:1-39.5x106) for high-risk hematologic malignancies between 1995 and 2012. Three out of 10 patients (33%) experienced GvHD early after HSCT; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. At a median follow-up of 7 years (range 2-14), all patients were in complete remission and free of GvHD, and displayed a complete and broad donor-derived immune system characterized by physiological counts of NK cells, B lymphocytes, γδ T cells and naïve and memory CD4+ or CD8+ T cells. TK cells were detected in all patients, at low levels (median=4cells/uL), even in patients treated with GCV. Ex vivo selection of pure TK-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Importantly, GCV sensitivity was preserved in long-term persisting TK cells, independently from their differentiation phenotype. Longitudinal follow up revealed that TK cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells. The low level of Ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. Polyclonality was demonstrated by sequencing among TK cells of thousands of diverse TCRs with a broad usage of V and J alpha and beta genes. The number of TK cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of TK cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Accordingly, we documented the persistence of CMV and Flu-specific TK cells only after post-transplant CMV reactivation or after Flu infection. Characterization of TK cell products infused to patients showed that the amount of infused TSCM cells positively correlates with early expansion and long-term persistence of gene-marked cells. By combining sorting of memory T-cell subsets with sequencing of integration sites, TCRα and TCRβ clonal markers, we longitudinally traced T-cell clones from infused products to late follow-up time-points. We showed that although T cells retrieved long-term are enriched in clones originally shared in different memory T-cell subsets, dominant long-term clonotypes preferentially originate from infused TSCM clones, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: In a completely restored immune system, suicide gene-modified donor T cells persist for up to 14 years in treated patients. Long-term persistence of memory T cells is determined by antigen exposure, and by the original phenotype of infused cells, according to a hierarchical model in which TSCM are superior to TCM and TEM/EFF. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Membership on an entity's Board of Directors or advisory committees. Bonini:MolMed S.p.A: Consultancy.

CD11b-Donor Dendritic Cells Elevate Donor T-Cell Production of Interferon-Gamma and Graft-Versus-Leukemia Activity in Allogeneic Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2181-2181
Author(s):  
Jian-Ming Li ◽  
Lauren Southerland ◽  
Cindy Giver ◽  
Doug McMillion ◽  
Wayn Harris ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Allogeneic Hsct ◽  
Graft Versus Leukemia ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Host antigen-presenting cells (APC) persist after high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) and initiate graft-versus-host disease (GvHD) in mouse models of HSCT. The role for donor APC on transplant outcomes is less clear. In clinical allogeneic HSCT from HLA-matched siblings, larger numbers of donor plasmacytoid dendritic cell (DC) precursors were associated with more relapse, and worse survival. Depletion of CD11b+ cells from bone marrow (containing CD11b+ DC) modestly augmented graft-versus-leukemia (GvL) activity in murine allogeneic HSCT. In this study, using allogeneic MHC mis-matched HSCT (C57BL/6→B10.BR) of mice bearing a lymphoblastic leukemia (LBRM), recipients of FACS–purified CD11b− donor DC plus FACS–purified HSC and T-cells had dramatically improved long-term survival (45% alive at >100 days) compared to d 5% survival among recipients of HSC and T-cells, or HSC, T-cells and CD11b+ DC (p<0.001). Both donor CD11b− and CD11b+ DC homed to lymphoid organs, and physically contacted donor T-cells, but only the CD11b− DC subset led to higher levels of interferon-γ (IFN- γ) in serum and higher levels of donor spleen-derived T-cells that synthesized IFN-γ in vivo in the first 10 days post-transplant. In contrast, recipients of CD11b+ DC had higher level of sera of IL–10. In CD11b− DC recipients, donor effector-memory CD8+ T-cells proliferated more rapidly and expanded in recipients, but did not cause debilitating GvHD in mice transplanted without leukemia. Donor-spleen-derived T-cells among recipients of CD11b− DC were predominately CD8+ (CD4:CD8 ratio 0.7:1), while donor spleen-derived T-cells were predominately CD4+ (p<0.02; CD4:CD8 ratio 1.3:1) among recipients of CD11b+ DC. In vitro co-culture of purified DC with homologous T-cells responding to allo-antigen demonstrated the same pattern of cytokine production as found in vivo, supporting the ability of CD11b− DC to generate Th1 immune responses that are associated with enhanced GvL effects and improved immune reconstitution. In vitro stimulation of DC subsets by CD40L (1μg/mL) and LPS (1μg/mL) showed that CD11b+ DC had higher level of PD–L1 expression compared to CD11b− DC. In vitro exposure of LBRM to IFN-γ, at doses of 10–300 pg/ml, similar to those observed in vivo, had no direct cytotoxic effect, and had no long-term growth-inhibitory effect on proliferation during 5 days of culture. CD11b+ DC expressed higher levels of PD–L1 and led to higher T-cell synthesis of IL–10 in vivo and in vitro. Transplantation of CD11b− DC resulted in higher levels of donor T-cell production of IFN-γ, and enhanced the GvL effect of donor T-cells without producing debilitating GvHD. These data indicated that purified donor DC can regulate post-transplant immunity via indirect antigen presentation to donor T-cells. CD11b+ DC expressed higher level of PD–L1 and led to higher level of IL–10 that resulted in more relapse and light GvHD. CD11b− DC led to higher level of donor IFN-γ that resulted in a stronger GvL effect while no debilitating GvHD. Engineering the DC content of an allograft may augment immune reconstitution and GvL activity without significantly increasing GvHD in allogeneic HSCT.

scholarly journals Regulation of specific cell-mediated cytotoxic response against SV40-induced tumor associated antigens by depletion of suppressor T cells with cyclophosphamide in mice.

1979 ◽  
Vol 149 (3) ◽  
pp. 774-779 ◽  
Author(s):  
M Glaser
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Effector T Cells ◽  
Specific Cell ◽  
51Cr Release ◽  
Tumor Associated Antigens ◽  
Specific Effector ◽  
Release Assay

When cyclophosphamide was administered to mice before immunization with syngeneic SV40 transformed cells, the specific immune response elicited, as was measured by in vitro 51Cr release assay was stronger and lasted longer when compared to the response generated in noncyclophosphamide-treated mice. The augmentation effect of the drug was dependent on cyclophosphamide concentration being optimal at 100 mg/kg and on the time of drug administration in relation to antigen immunization being optimal at 2 d before antigen administration. Transfer of T cells from normal syngeneic mice to drug-treated animals abolished the cyclophosphamide-induced augmentation of immune response. These results implied that cyclophosphamide sensitive T cells suppressed the in vivo generation of specific effector T cells against SV40-induced tumor-associated antigens.

scholarly journals 323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A348-A348
Author(s):  
Jessie Wang ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Chenpan Nie ◽  
Annie An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Evasion ◽  
Syngeneic Mouse ◽  
Ifn Γ ◽  
Draining Lymph Nodes ◽  
Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

Escape from Suppression: Tumor-Specific Effector Cells Out-Compete Regulatory T Cells Following Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 69-69
Author(s):  
Paria Mirmonsef ◽  
Gladys Tan ◽  
Gang Zhou ◽  
Tricia Pyhel ◽  
Ivan M. Borrello ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Tumor Antigen ◽  
Immune Reconstitution ◽  
Effector T Cells ◽  
Transplant Recipients ◽  
Tumor Bearing ◽  
Established Tumor ◽  
Specific Effector

Abstract Autologous hematopoietic stem cell (HSC) transplantation is an accepted therapy for many hematological malignancies. High dose chemo-radiation reduces tumor burden but also ablates lymphohematopoiesis. Subsequent infusion of cellular grafts containing HSC and mature lymphocytes “rescues” the host from this otherwise lethal ablation, and initiates immune reconstitution. In many systems, tumor-specific T cells are functionally tolerant in the presence of established tumor. Paradoxically, however, the infusion of these lymphocytes into irradiated tumor-bearing syngeneic recipients unmasks effector function manifested as prolonged progression-free survival when compared to recipients treated with lymphocytes from non-tumor bearing donors. We have recently demonstrated that this tolerant tumor-specific T cell population from mice with established tumor is in fact a heterogeneous mixture of naive, effector, and regulatory T cells (Tregs), which as a whole are rendered functionally unresponsive through dominant suppression. The apparent reversal of tolerance in the post-transplant setting prompted a more detailed examination of the fate of these individual components during immune reconstitution. Here, we show that CD4+ T cells specific for a model tumor antigen are hyporesponsive to antigen when isolated from mice harboring an established systemic B cell lymphoma. Upon transfer into irradiated lymphoma-bearing mice, however, these cells undergo robust antigen-driven clonal expansion, and their ability to produce interferon gamma (IFNγ) is restored. Notably, in spite of the presence of tumor in the transplant recipients, tolerance to tumor antigen was not established in the early post-transplant period, even for mice receiving naive T cells in the graft. Tumor-specific CD4+CD25+Foxp3+ Tregs isolated from the donors were found to undergo a modest tumor-antigen-driven expansion in transplant recipients. When isolated from recipients, such cells maintained expression of Foxp3 and their capacity to suppress naive T cells when cultured in vitro. However, the presence of tumor-specific Tregs failed to significantly inhibit the expansion of naive or effector T cells specific for tumor in vivo, when examined 2 weeks post BMT. Indeed, the expansion of tumor-specific effector T cells significantly exceeded the expansion of Tregs, resulting in a nearly five-fold increase in the effector:Treg ratio. At the ratios present during this phase of immune reconstitution, the frequency of Tregs was insufficient to suppress effector cell function (proliferation and IFNγ production) when studied in vitro. This accounts for the reversal of tolerance identified in the population as a whole and its capacity to mediate tumor rejection.

CD11b− Donor Dendritic Cells Enhance Donor T-Cells’ Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1252-1252 ◽  
Author(s):  
Jian-Ming Li ◽  
Kataryna A. Darlak ◽  
Ying Lu ◽  
Cynthia Giver ◽  
Wayne Harris ◽  
...  
Keyword(s):  
T Cells ◽  
Serum Levels ◽  
Hematopoietic Stem ◽  
Treatment Groups ◽  
Post Transplant ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Th1 Cytokines

Abstract Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007). Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT). Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients’ peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure. Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p<0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect. Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

Safely Improving the in Vivo Survival of Tumor Specific Cytotoxic T Lymphocytes by Co-Transfer of IL7 Receptor Alpha Chain and icaspase9

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3534-3534
Author(s):  
Juan F Vera ◽  
Valentina Hoyos ◽  
Barbara Savoldo ◽  
Concetta Quintarelli ◽  
Greta A Giordano ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Adoptive Transfer ◽  
Fold Increase ◽  
Suicide Gene ◽  
Antigen Specificity ◽  
Anti Tumor Activity

Abstract Providing a proliferative and survival advantage to tumor-specific cytotoxic T lymphocytes (CTLs) remains a challenge in the adoptive therapy of cancer patients. It is now evident that the in vivo expansion of T cells after adoptive transfer is best accomplished in the lymphodepleted host due to the increased production of endogenous IL15 and IL7, which help restore lymphopoiesis. We have found that antigen activated cytotoxic T lymphocytes (CTLs) directed to tumor associated epitopes (for example derived from EBV, or from cancer testis antigens such as PRAME) down regulate a chain of IL7R, a common γ chain cytokine receptor, impairing their capacity to respond to IL7. We hypothesized that despite receptor downregulation, the signal transduction pathway for IL7R would remain intact in the CTLs so that forced expression of IL7Rα would restore IL7 responsiveness and improve in vivo expansion and survival of CTLs. We used EBV-specific CTLs as our model, and showed in vitro that a functional IL-7Ra molecule can be expressed in CTLs using retroviral gene transfer so that the percentage of receptor + cells increased from 2.4%±0.5% to 50%±20. This modification restored the in vitro proliferation of genetically modified CTLs in response to IL7 so that cell numbers increased from 1×106 cells to 0.1×109 (range, 0.6×108 to 0.3×109)] comparable with the effects of IL2 [from 1×106 cells to 0.7×109 (range, 0.7×107 to 1.6×109)] In contrast, control EBV-CTL with IL7 progressively declined in number (p<0.001) These effects were accomplished without alteration of antigen specificity or responsiveness to other common γ chain cytokines, and cell survival remained antigen dependent. In a xenogeneic mouse model, CTLs expressing IL7Ra significantly expanded in vivo in response to EBV-tumor antigen and the administration of IL7. By day 15, both control CTLs and IL7Ra+ CTLs had modestly proliferated in response to IL-2 (2.3 fold, range 1.1–5.1 for control CTLs, and 2.67 fold, range 0.6 to 8.15 for IL7Ra+ CTLs). In contrast, only IL7Ra+ CTLs significantly expanded in the presence of IL7, showing a 6.09 fold increase (range 0.7 to 25.2) compared to mice that received control CTLs and IL7 (0.9 fold, range 0.5–1.7) (p<0.0001). Modified CTLs also provided enhanced anti-tumor activity. SCID mice engrafted i.p with 3×106 tumor cells marked with Firefly luciferase, showed a rapid increase in signal in the absence of CTLs (Fold increase in luminance = 29.8 median, range 4.4 to 103) by day 14 after tumor engraftment. Similar tumor growth was observed in mice receiving IL7Ra+ CTLs without cytokines (luminance increase14.4 fold, range 1 to 90). In contrast, mice receiving IL7Ra+ CTLs and either IL2 or IL7, had a decline in tumor luminance (fold expansion 0.7, range 0.08 to 2.9, and 0.8, range 0.004 to 3.5, respectively p<0.0001). Although growth of the transgenic T cells remained antigen dependent, as a further safety measure, we incorporated an inducible suicide gene based on icaspase9 that can be activated by exposure to a small chemical inducer of dimerization (CID) (AP20187). Incorporation of this suicide gene did not affect the in vitro or in vivo anti-tumor activity of the CTL’s but allowed them to be rapidly eliminated. So that after a single dose of CID (50 nM) the transgenic population were decreased by >98.5% We conclude that forced expression of the IL-7Ra by CTLs can be used to recapitulate the response of these cells to this cytokine and thereby promote their in vivo anti-tumor activity after adoptive transfer either in a lymphodepleted host or after the administration of the recombinant protein.

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