Ag-specific Cytotoxic T Lymphocytes Research Articles

Tumorous expression of NAC1 restrains antitumor immunity through the LDHA-mediated immune evasion

BackgroundT cell-mediated antitumor immunity has a vital role in cancer prevention and treatment; however, the immune-suppressive tumor microenvironment (TME) constitutes a significant contributor to immune evasion that weakens antitumor immunity....

Upregulation of CD3ζ and L‐selectin in antigen‐specific cytotoxic T lymphocytes by eliminating myeloid‐derived suppressor cells with doxorubicin to improve killing efficacy of neuroblastoma cells in vitro

BackgroundAgglomeration of myeloid‐derived suppressor cells (MDSCs) in tumors impedes immunotherapeutic effects. Doxorubicin (DOX) is currently the most specific drug used for the selective removal of MDSCs. Here, we study the feasibility and mechanism of eliminating MDSCs by DOX to improve antigen‐specific cytotoxic T lymphocyte (CTL)‐killing neuroblastoma (NB) cells in vitro.MethodsCTL and MDSC were prepared; then, CTLs, NB cells, MDSCs, and DOX were mixed and cultivated in different collocation patterns and divided into different groups. The levels of cluster of differentiation 3ζ chain (CD3ζ) and L‐selectin in CTL in different groups were detected. Thereafter, the killing rate of NB cells and secretion of interleukin‐2 and interferon‐γ were measured and compared.ResultsBy real‐time polymerase chain reaction (PCR) and Western blot test respectively, the proliferation and killing effect of CTLs on NB cells were all inhibited by MDSC through downregulating CD3ζ (p = 0.002; p = 0.001) and L‐selectin (p = 0.006; p < 0.001). However, this inhibitory effect was reversed by DOX. Significant differences were observed in the levels of interleukin‐2 (p < 0.001), interferon‐γ (p < 0.001), and the killing rate (p < 0.001) among the groups, except between the CTL +SK‐N‐SH and CTL +SK‐N‐SH +DOX groups (p > 0.05).ConclusionsTargeted elimination of MDSCs by DOX can improve Ag‐specific CTL killing of NB cells in vitro by upregulating CD3ζ and L‐selectin. This study provides a novel method to enhance the immunotherapeutic effects of NB.

Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HIV Replication and PD-1 Expression on CD4+ T Cells.

The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the human immunodeficiency virus (HIV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the local tissues to suppress HIV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs elicit the antiviral response remain to be fully elucidated. In this study, we generated the functional HIV-1 Gag epitope SL9-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the suppression of SL9-specific iPSC-CTLs on viral replication and the protection of CD4+ T cells. A chimeric HIV-1, i.e., EcoHIV, was used to produce HIV replication in mice. We show that adoptive transfer of SL9-specific iPSC-CTLs greatly suppressed EcoHIV replication in the peritoneal macrophages and spleen in the animal model. Furthermore, we demonstrate that the adoptive transfer significantly reduced expression of PD-1 on CD4+ T cells in the spleen and generated persistent anti-HIV memory T cells. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the local tissues to suppress HIV replication and prevent CD4+ T cell exhaustion through reduction of PD-1 expression.

Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HBV Replication through Production of IFN-γ and TNF-⍺.

SummaryThe viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed HBV replication and prevented HBV surface Ag expression. We further demonstrate that the adoptive transfer significantly increased T cell accumulation and production of antiviral cytokines. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the liver and suppress HBV replication through producing antiviral cytokines.

Suppression of HBV replication in mice using stem cell-derived viral antigen-specific T lymphocytes

Abstract The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV surface Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model. A hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver induced HBV replication in mice. We show that adoptive transfer of HBV surface Ag-specific iPSC-CTLs greatly suppressed the HBV replication in the liver and blood in the animal model and prevented the HBV surface Ag expression on hepatocytes. Furthermore, we demonstrate that the adoptive transfer significantly increased the migration of CD8+ T cells and the production of the antiviral cytokines (IFN-g, TNF-a) in the liver. These results indicate that the stem cell-derived tissue-associated CTLs can robustly accumulate in the liver and suppress the HBV replication through producing antiviral cytokine.

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

The Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HBV Replication Through Production of IFN-γ and TNF-⍺

The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the hepatitis B virus (HBV) infection. After adoptive transfer, PSC-CTLs can be programmed to be HBV-specific and to infiltrate into the liver to suppress HBV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs provoke the antiviral response remain to be fully elucidated. In this study, we generated the functional HBV Ag-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the underlying mechanisms of the CTL-mediated antiviral replication in a murine model of HBV infection. A hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver induced HBV replication efficiently in mice. We show that adoptive transfer of HBV Ag-specific iPSC-CTLs greatly suppressed the HBV replication in the liver and blood in the animal model and prevented the HBV surface Ag expression on hepatocytes. We further demonstrate that the adoptive transfer significantly increased the migration of CD8+ T cells and the production of the antiviral cytokines (IFN-γ, TNF-α) in the liver. Moreover, using neutralizing antibody to IFN-r or/and TNF-a dramatically dismissed the suppression of the HBV replication mediated by the HBV Ag-specific iPSC-CTLs. These results indicate that the stem cell-derived tissue-associated CTLs can robustly accumulate in the liver and suppress the HBV replication through producing the antiviral cytokines IFN-r and TNF-a.

Monocytes outperform ex vivo generated dendritic cells as cellular vaccines to trigger cytotoxic T lymphocyte responses against cancer in pre-clinical models

Abstract Conventional dendritic cell (DC) vaccines using ex vivo generated DC have been widely tested in clinical trials in the treatment of human cancers. These DC vaccines have often displayed limited efficacy despite a complex manufacturing process. Although the efficacy of DC vaccines is generally thought to depend on their antigen-presenting cell function, several lines of evidence suggest that, instead of directly presenting antigen (Ag) to naïve T cells, administered DC trigger cytotoxic T lymphocyte (CTL) responses in vivo mainly by transferring Ag to lymphoid-resident conventional DC (cDC). We tested here whether monocytes, known to transfer Ag to cDC in vivo, can be directly loaded with tumor Ag to trigger robust anti-tumor CTL responses. We found that IV administered monocytes loaded with protein or peptide Ag triggered stronger Ag-specific CTL responses than conventional adjuvant-based and DC vaccines. In several murine cancer models, including intra-cranial glioblastoma, we showed that monocyte vaccination suppressed tumor growth better than conventional adjuvant-based, GVAX and DC vaccines. Such anti-tumor activities could be further enhanced with immune checkpoint inhibition. Ag-loaded monocytes did not directly activate naïve CD8+T cells. Instead, they formed stable physical contacts with and transferred Ag to splenic CD8+cDC. This Ag transfer required the formation of connexin 43-containing gap junctions between monocytes and CD8+cDC. Our findings demonstrated that gap junctions are an efficient Ag transfer pathway in vivo between monocytes and splenic CD8+cDC and suggested that IV administered Ag-loaded monocytes may serve as a simple and efficacious cellular vaccine platform for the treatment of human cancers.

Optimized Gemcitabine Therapy in Combination with E7 Peptide Immunization Elicits Tumor Cure by Preventing Ag-Specific CTL Inhibition in Animals with Large Established Tumors.

The role of chemotherapeutic agents in tumor immunotherapy is still controversial. In this study, we test using a TC-1 tumor model whether gemcitabine plus E7 peptide vaccine regimens (E7 peptides+CpG-ODN+anti-4-1BB Abs) may result in tumor cure in mice with large established tumors, with a focus on their effects on Ag-specific cytotoxic T lymphocyte (CTL) and myeloid-derived suppressor cell levels. Gemcitabine inhibited tumor growth by its direct cytotoxicity to tumor cells in vivo. E7 peptide vaccine regimens enhanced Ag-specific CTL lytic and antitumor therapeutic activity. Initial combination therapy using gemcitabine and E7 peptide vaccine regimens resulted in tumor regression with tumor relapse in animals with large established tumors, which appeared to result from the suppression of Ag-specific CTL activity by gemcitabine treatment. However, optimization of gemcitabine therapy by reducing its dose and frequency led to complete tumor regression without any recurring tumors in all tested mice even after discontinuation of therapy, possibly due to Ag-specific CTL responses. Thus, this study shows that the optimal dose and therapy frequency of gemcitabine are critical for achieving tumor cure in tumor-bearing animals undergoing E7 peptide vaccine regimen therapy, mainly by preventing CTL suppression. These findings may have implications for designing peptide-based therapeutic vaccines in cancer patients undergoing chemotherapy.

Protective Cancer Vaccine Using Genetically Modified Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) yield both the myeloid and lymphoid lineages of blood cells and can be reprogrammed into tumor antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) to prevent tumor growth. However, the optimal approach for differentiating tumor Ag-specific CTLs from HSCs, such as HSC-CTLs, remains elusive. In the current study, we showed that a combination of genetic modification of HSCs and in vivo T cell development facilitates the generation of Ag-specific CTLs that suppressed tumor growth. Murine HSCs, which were genetically modified with chicken ovalbumin (OVA)-specific T cell receptor, were adoptively transferred into recipient mice. In the following week, mice were administered with intraperitoneal injections of an agonist α-Notch 2 antibody and cytokines (rFlt3L and rIL-7) three times. After another two weeks, mice received a subcutaneous inoculation of B16-OVA melanoma cells that express OVA as a surrogate tumor Ag, before the anti-tumor activity of HSC-derived T cells was assessed. OVA-specific CTLs developed in vivo and greatly responded to OVA Ag stimulation ex vivo. In addition, mice receiving genetically modified HSCs and in vivo priming established anti-tumor immunity, resulting in the suppression of tumor growth. These results reported in this present study provide an alternative strategy to develop protective cancer vaccines by using genetically modified HSCs.

The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells.

The TLR2 agonist, dipalmitoyl lipopeptide (Pam2LP), has been used as an immune adjuvant without much success. Pam2LP is recognised by TLR2/6 receptors in humans and in mice. This study examined the proliferative activity of cytotoxic T lymphocytes (CTL) using mouse Ag-presenting dendritic cells (DCs) and OT-I assay system, where a library of synthetic Pam2LP was utilised from the Staphylococcus aureus database. Ag-specific CTL expansion and IFN-γ levels largely depended on the Pam2LP peptide sequence. The first Aa is cysteine (Cys), which has an active SH residue to bridge fatty acids, and the second and third Aa are hydrophilic or non-polar. The sequence structurally adapted to the residual constitution of the reported TLR2/6 pocket. The inactive sequence contained proline or leucine/isoleucine after the first Cys. Notably, no direct activation of OT-I cells was detected without DCs by stimulation with the active Pam2LP having the Cys-Ser sequence. MyD88, but not TICAM-1 or IFN pathways, in DCs participates in DC maturation characterised by upregulation of CD40, CD80 and CD86. Hence, the active Pam2LPs appear suitable for dimeric TLR2/6 on DCs, resulting in induction of DC maturation.

T Helper 17 Promotes Induction of Antigen-Specific Gut-Mucosal Cytotoxic T Lymphocytes following Adenovirus Vector Vaccination.

Few current vaccines can establish antigen (Ag)-specific immune responses in both mucosal and systemic compartments. Therefore, development of vaccines providing defense against diverse infectious agents in both compartments is of high priority in global health. Intramuscular vaccination of an adenovirus vector (Adv) has been shown to induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut-mucosal compartments. We previously found that type I interferon (IFN) signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that T helper 17 (Th17)-polarizing cytokine expression was down-regulated in the inguinal lymph nodes (iLNs) of Ifnar2−/− mice, resulting in the reduction of Ag-specific Th17 cells in the iLNs and gut mucosa of the mice. We also found that prior transfer of Th17 cells reversed the decrease in the number of Ag-specific gut-mucosal CTLs in Ifnar2−/− mice following Adv vaccination. Additionally, prior transfer of Th17 cells into wild-type mice enhanced the induction of Ag-specific CTLs in the gut mucosa, but not in systemic compartments, suggesting a gut mucosa-specific mechanism where Th17 cells regulate the magnitude of vaccine-elicited Ag-specific CTL responses. These data suggest that Th17 cells translate systemic type I IFN signaling into a gut-mucosal CTL response following vaccination, which could promote the development of promising Adv vaccines capable of establishing both systemic and gut-mucosal protective immunity.

Development and characterization of naive single-type tumor antigen-specific CD8+ T lymphocytes from murine pluripotent stem cells

ABSTRACTOptimal approaches to differentiate tumor antigen-specific cytotoxic T lymphocytes (CTLs) from pluripotent stem cells (PSCs) remain elusive. In the current study, we showed that combination of in vitro priming through Notch ligands and in vivo development facilitated the generation of tumor Ag-specific CTLs that effectively inhibited tumor growth. We co-cultured the murine induced PSCs (iPSCs) genetically modified with tyrosinase-related protein 2 (TRP2)-specific T cell receptors with OP9 cell line expressing both Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4) for a week before adoptively transferred into recipient C67BL/6 mice. Three weeks later, B16 melanoma cells were inoculated subcutaneously, and the antitumor activity of the iPSC-derived T cells was assessed. We observed the development of the TRP2-specific iPSC-CD8+ T cells that responded to Ag stimulation and infiltrated into melanoma tissues, significantly inhibited the tumor growth, and improved the survival of the tumor-bearing mice. Thus, this approach may provide a novel effective strategy to treatment of malignant tumors.

Dissection of CD8+ T cell immunodominance hierarchy in human influenza virus infection

Abstract Based on magnitude, virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses can be grouped as immunodominant and subdominant. Such immunodominance hierarchies are governed by multiple factors, including naïve T cell precursor frequency, proliferative capacity and clonotypic diversity of Ag-specific CTLs. However, how these factors determine together inter/intra-individual immunodominance remains elusive. In this study, we dissected CTL immunodominance hierarchy for universally conserved influenza (IAV)-specific epitopes. Our data from 59 donors covering the HLAI types presenting universally conserved IAV-specific peptides, HLA-A* 0201, A*0301, B*2705, B*5701, B*1801 and B*0801, revealed that CTLs directed against HLA-A* 0201:M158 were immunodominant, except in A*0201+B*2705+ heterozygous individuals, where the HLA-B* 2705-NP383 recalls higher magnitude CTL responses. Subsequently, we further dissected the basis of B*2705 immundomination over A*0201 by analyses of T cell Receptor (TCRαβ) repertoire, TCR avidity and kinetics of killing and proliferation. Our data show that following in vitro peptide stimulation, B*2705:NP383 CTLs proliferate rapidly acquiring higher levels of polyfunctionality than A*02:M158 specific CTLs. Furthermore, ex vivo single-cell paired CDR3αβ analysis revealed that public A*0201:M158 TCRs characterized by Vβ19/Vα27 profiles in A*0201 individuals are significantly reduced/absent in A*0201+B*2705+ individuals. Clonal analysis of M158 dominant public TCRs suggested B*2705-mediated removal of self-reactive TCRs and is under investigation. Our study provides insights into the importance of human IAV-specific immunodominance at quantitative and qualitative levels.

Targeting stem cell-derived viral antigen-specific T lymphocytes for HBV immunotherapy

Abstract Adoptive cell transfer (ACT) of hepatitis B virus (HBV)-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for chronic HBV infection. Naive or central memory T cell-derived effector CTLs known as “highly reactive” cells are optimal populations for cell-based therapies. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of CTLs from patients. Pluripotent stem cells (PSCs) have the capacity to be reprogrammed into functional viral antigen (Ag)-specific CTLs, which have a potential to be used for ACT-based immunotherapy. However, the optimal approach to differentiate viral Ag -specific CTLs from PSCs remains unclear. In this study, we generated HBV-specific CTLs from induced PSCs (iPSCs), i.e., iPSC-CTLs, and utilized a mouse model of chronic HBV infection to test ACT of iPSC-CTLs for HBV immunotherapy. Murine iPSCs that genetically modified with HBV-specific T cell receptor (TCR) were co-cultured with OP9 cell line expressing Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4 cell) for a week and adoptively transferred into recipient C67BL/6 mice for in vivo development. The combination of Notch signaling and incorporation of HBV-specific TCR genes in iPSCs drove the development of HBV-specific iPSC-CTLs. In addition, In a mouse model of chronic HBV infection in which the HBV viral genome is delivered into the mouse liver through hydrodynamic injection, HBV-specific iPSC-CTLs infiltrated into liver tissue, and inhibited HBV replication after ACT. These results confirm that ACT of viral Ag-specific iPSC-CTLs can substantially suppress HBV replication, and indicate that stem cell-derived highly reactive viral Ag-specific CTLs may be used for HBV immunotherapy.

Programmed Death-Ligand 1 on Antigen-presenting Cells Facilitates the Induction of Antigen-specific Cytotoxic T Lymphocytes: Application to Adoptive T-Cell Immunotherapy.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

Melanoma Immunotherapy in Mice Using Genetically Engineered Pluripotent Stem Cells.

Adoptive cell transfer (ACT) of antigen (Ag)-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a highly promising treatment for a variety of diseases. Naive or central memory T-cell-derived effector CTLs are optimal populations for ACT-based immunotherapy because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells, and have the higher ability to respond to homeostatic cytokines. However, such ACT with T-cell persistence is often not feasible due to difficulties in obtaining sufficient cells from patients. Here we present that in vitro differentiated HSCs of engineered PSCs can develop in vivo into tumor Ag-specific naive CTLs, which efficiently suppress melanoma growth. Mouse-induced PSCs (iPSCs) were retrovirally transduced with a construct encoding chicken ovalbumin (OVA)-specific T-cell receptors (TCRs) and survival-related proteins (i.e., BCL-xL and survivin). The gene-transduced iPSCs were cultured on the delta-like ligand 1-expressing OP9 (OP9-DL1) murine stromal cells in the presence of murine recombinant cytokines (rFlt3L and rIL-7) for a week. These iPSC-derived cells were then intravenously adoptively transferred into recipient mice, followed by intraperitoneal injection with an agonist α-Notch 2 antibody and cytokines (rFlt3L and rIL-7). Two weeks later, naive OVA-specific CD8(+) T cells were observed in the mouse peripheral lymphatic system, which were responsive to OVA-specific stimulation. Moreover, the mice were resistant to the challenge of B16-OVA melanoma induction. These results indicate that genetically modified stem cells may be used for ACT-based immunotherapy or serve as potential vaccines.

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

UV irradiation of immunized mice induces type 1 regulatory T cells that suppress tumor antigen specific cytotoxic T lymphocyte responses

We previously showed that exposure to UV radiation after immunization suppresses Th1 and Th2 immune responses, leading to impaired Ab and allo-immune responses, but the impact of UV radiation after immunization on anti-tumor immune responses mediated by tumor-specific CD8(+) T cell responses remains less clear. Furthermore, the exact phenotypic and functional characteristics of regulatory T cell population responsible for the UV-induced immunosuppression still remain elusive. Using the MBL-2 lymphoma cell line engineered to express OVA as a surrogate tumor Ag, here we demonstrate that UV irradiation after tumor Ag-immunization suppresses the anti-tumor immune response in a manner dependent on the immunizing Ag. This suppression was mediated by interleukin (IL)-10 released from CD4(+) CD25(+) T cells, by which impaired the induction of cytotoxic T lymphocytes (CTL) able to kill Ag-expressing tumor cells. In addition, we generated a panel of T cell clones from UV-irradiated and non-irradiated mice, and all of the clones derived from UV-irradiated mice had a Tr1-type regulatory T cell phenotype with expression of IL-10 and c-Maf, but not Foxp3. These Tr1-type regulatory T cell clones suppressed tumor rejection in vivo as well as Th cell activation in vitro in an IL-10 dependent manner. Given that suppression of Ag-specific CTL responses can be induced in Ag-sensitized mice by UV irradiation, our results may imply that exposure to UV radiation during premalignant stage induces tumor-Ag specific Tr1 cells that mediate tumor-Ag specific immune suppression resulting in the promotion of tumor progression.