Antigen-specific Cytotoxic T Lymphocytes Research Articles

Development of Cell Technologies Based on Dendritic Cells for Immunotherapy of Oncological Diseases.

Immunotherapy using dendritic cell-based vaccination is a natural approach using the capabilities and functions inherent in the patient's immune system to eliminate tumor cells. The development of dendritic cell-based cell technologies evolved as the disorders of dendritic cell differentiation and function in cancer were studied; some of these functions are antigen presentation, priming of cytotoxic T-lymphocytes and induction of antigen-specific immune responses. At the initial stage of technology development, it was necessary to develop protocols for the in vitro generation of functionally mature dendritic cells that were capable of capturing tumor antigens and processing and presenting them in complex with MHC to T-lymphocytes. To achieve this, various forms of tumor-associated antigen delivery systems were tested, including lysates, tumor cell proteins (peptides), and DNA and RNA constructs, and it was shown that the use of DNA and RNA constructs was the most effective method, as it made it possible not only to deliver the most immunogenic epitopes of tumor-associated antigens to dendritic cells, but also to enhance their ability to induce antigen-specific cytotoxic T-lymphocytes. Currently, cell therapy based on dendritic cells is a modern basis for antigen-specific immunotherapy of cancer due to the simplicity of creating DNA and RNA constructs encoding information about both target tumor antigens and regulatory molecules. The potential development of cell technologies based on dendritic cells aims to obtain antigen-specific cytotoxic T-lymphocytes induced by dendritic cells, study their functional activity and develop cell-based therapy.

Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor-positive Breast Cancer.

Estrogen receptor (ESR1) mutations have emerged as a key factor in endocrine therapy resistance. We identified and validated five novel, immunogenic ESR1-derived peptides that could be targeted through vaccine-based immunotherapy.

Use of Stromal Intervention and Exogenous Neoantigen Vaccination to Boost Pancreatic Cancer Chemo-Immunotherapy by Nanocarriers.

Despite the formidable treatment challenges of pancreatic ductal adenocarcinoma (PDAC), considerable progress has been made in improving drug delivery via pioneering nanocarriers. These innovations are geared towards overcoming the obstacles presented by dysplastic stroma and fostering anti-PDAC immune reactions. We are currently conducting research aimed at enhancing chemotherapy to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD). This is accomplished using lipid bilayer-coated nanocarriers, which enable the attainment of synergistic results. Noteworthy examples include liposomes and lipid-coated mesoporous silica nanoparticles known as "silicasomes". These nanocarriers facilitate remote chemotherapy loading, as well as the seamless integration of immunomodulators into the lipid bilayer. In this communication, we elucidate innovative ways for further improving chemo-immunotherapy. The first is the development of a liposome platform engineered by the remote loading of irinotecan while incorporating a pro-resolving lipoxin in the lipid bilayer. This carrier interfered in stromal collagen deposition, as well as boosting the irinotecan-induced ICD response. The second approach was to synthesize polymer nanoparticles for the delivery of mutated KRAS peptides in conjunction with a TLR7/8 agonist. The dual delivery vaccine particle boosted the generation of antigen-specific cytotoxic T-cells that are recruited to lymphoid structures at the cancer site, with a view to strengthening the endogenous vaccination response achieved by chemo-immunotherapy.

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immuneresponse of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic Tcell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Regulating T-cell metabolic reprogramming and blocking PD-1 co-promote personalized postoperative autologous nanovaccines

Cytotoxic T lymphocytes (CTLs) are central effector cells in antitumor immunotherapy. However, the complexity of immunosuppressive factors in the immune system contributes to the low response rates of current CTL-based immunotherapies. Here, we propose a novel holistic strategy including a priming response, promoting activity, and relieving suppression of CTLs, aiming to strengthen the effect of personalized postoperative autologous nanovaccines. The nanovaccine (C/G-HL-Man) fused the autologous tumor cell membrane with dual adjuvants (CpG and cGAMP), and could effectively accumulate in lymph nodes and promote antigen cross presentation by dendritic cells to prime a sufficient specific-CTL response. The PPAR-α agonist fenofibrate was used to regulate T-cell metabolic reprogramming to promote antigen-specific CTLs activity in the harsh metabolic tumor microenvironment. Finally, the PD-1 antibody was used to relieve the suppression of specific-CTLs in the immunosuppressive tumor microenvironment. In vivo, the C/G-HL-Man exhibited strong antitumor effect in the B16F10 murine tumor prevention model and the B16F10 postoperative recurrence model. In particular, combination therapy with nanovaccines, fenofibrate, and PD-1 antibody effectively inhibited the progression of recurrent melanoma and prolonged the survival time. Our work highlights the critical role of the T-cell metabolic reprogramming and PD-1 blocking in autologous nanovaccines, offering a novel strategy for strengthening the function of CTLs.

Exosomes from Ub‑HBcAg‑overexpressing dendritic cells induce T‑lymphocyte differentiation and enhance cytotoxic T‑lymphocyte activity.

Hepatitis B virus (HBV) infection is a major public health concern. The clearance of HBV may involve cytotoxic T-lymphocyte (CTL) activity and T helper type 1 reactions. Exosomes generated from dendritic cells (DCs) can induce immunological responses capable of eradicating viruses. However, exosomes loaded with antigens have not yet demonstrated therapeutic potential in HBV infection. Therefore, the present study aimed to investigate the antiviral effects of DC-derived exosomes (Dexs) loaded with ubiquitinated HBV core antigen (Dexs-Ub-HBcAg). Murine bone marrow-derived DCs were loaded with a recombinant lentivector encoding the ubiquitinated form of HBcAg. High-purity Dexs were generated using differential velocity centrifugation. Splenic T-lymphocytes were stimulated with Dexs-Ub-HBcAg and the specific T-cell-mediated immune responses were examined. Cytokine expression was analyzed using enzyme-linked immunosorbent assays. T-lymphocyte proliferation was detected using a Cell Counting Kit-8 assay and HBcAg-specific CTL activity was determined using a lactate dehydrogenase release assay. The results revealed that Dexs-Ub-HBcAg effectively stimulated T-cell proliferation and induced the activation of antigen-specific CTLs to exhibit HBcAg-specific CTL immune responses in vitro. These results suggest the potential of Dexs-Ub-HBcAg for development as a future therapeutic option for the elimination of HBV.

Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells

BackgroundImmune tolerance contributes to resistance to conventional cancer therapies such as radiation. Radiotherapy induces immunogenic cell death, releasing a burst of tumor antigens, but this appears insufficient to stimulate an...

CD200+ cytotoxic T lymphocytes in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy.

Anti-PD-1/PD-L1 therapy, either by anti-PD-1 antibody or anti-PD-L1 antibody, has efficacy by reinvigorating tumor-infiltrating CD8+ T cells in a subset of patients with cancer, but it has unequal effects on heterogeneous CD8+ T cell populations. Hence, the subset crucial to efficacious PD-1 blockade therapy remains elusive. Here, we found an increase in tumor-infiltrating CD200+ cytotoxic T lymphocytes (CTLs) upon PD-1/PD-L1 blockade, with higher proportions of CD200+ T cells positively related to a favorable clinical outcome to anti-PD-1/PD-L1 therapy in three independent cohorts of patients with cancer. Using multiple mouse tumor models, we demonstrated that CD200+ CTLs are essential for efficacious anti-PD-L1 therapy. Mechanistically, we observed a unique chromatin landscape in CD200+ CTLs and found that these cells are enriched for tumor antigen-specific CTLs and have antitumor effector functions. Coinoculation of CD200+ CTLs with tumor cells led to robust tumor regression in two transplanted mouse models. Clinically, we found that infiltration of CD200+ CTLs into tumors could predict immunotherapy efficacy in six patient cohorts. Together, our findings reveal that CD200+ CTLs in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy and could serve as a predictor of successful immunotherapy in the clinic.

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic Tcell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for Tcell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ Tcell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

Regulatory T-Cells Suppress Cytotoxic T Lymphocyte Responses against Microglia.

Regulatory T-cells (Tregs) play pivotal roles during infection, cancer, and autoimmunity. In our previous study, we demonstrated a role for the PD-1:PD-L1 pathway in controlling cytolytic responses of CD8+ T lymphocytes against microglial cells presenting viral peptides. In this study, we investigated the role of Tregs in suppressing CD8+ T-cell-mediated cytotoxicity against primary microglial cells. Using in vitro cytotoxicity assays and flow cytometry, we demonstrated a role for Tregs in suppressing antigen-specific cytotoxic T-lymphocyte (CTL) responses against microglia loaded with a model peptide (SIINFEKL). We went on to show a significant decrease in the frequency of IFN-γ- and TNF-producing CD8+ T-cells when cultured with Tregs. Interestingly, a significant increase in the frequency of granzyme B- and Ki67-producing CTLs was observed. We also observed a significant decrease in the production of interleukin (IL)-6 by microglia. On further investigation, we found that Tregs significantly reduced MHC class 1 (MHC-1) expression on IFN-γ-treated microglial cells. Taken together, these studies demonstrate an immunosuppressive role for Tregs on CTL responses generated against primary microglia. Hence, modulation of Treg cell activity in combination with negative immune checkpoint blockade may stimulate anti-viral T-cell responses to more efficiently clear viral infection from microglial cell reservoirs.

Symposium 2-2 - Challenges in elucidating the mechanisms of fetal recognition and tolerance by T cells

The fetus is a semi-graft expressing paternally derived antigens that are foreign to the mother, however, it is not rejected by the mother because of the establishment of feto-maternal tolerance. Maternal cytotoxic T cells (CTLs) and CD4+ T helper cells (CD4+Tconv) recognize fetal antigens at the T cell receptor (TCR), while regulatory T cells (Treg) suppress immune responses. To investigate the role of antigen-specific Tregs and CTLs in human pregnancy, we analyzed the TCR variations from single T cells from decidua. In normal pregnancies, the number of Tregs with specific TCRs which were presumed to be paternal antigen-specific Tregs increased in a biased manner. In preeclampsia, antigen specific Tregs were decreased. This suggests that induction of antigen-specific immune tolerance by Tregs was inadequate in preeclampsia. Next, to comprehensively search for pathologically relevant molecules in Tregs and CD4+Tconv that could be targets for immunological therapy, we performed single-cell transcriptome analysis of T cells. The results suggest that Th17 among CD4+Tconvs is increased in PE, but the differentiation pathway may be different from that of Tregs. We would like to mention the results of this study and the prospects for immunological approaches to PE therapy.

Educational Lecture 2 - Potential of Kampo medicine in cancer immunotherapy

Cancer immunosurveillance is an intrinsic defense mechanism in which out host immune responses suppress cancer development. As a result of such immunological surveillance system, host immune response also edits cancer cells which is called as cancer immunoediting process. Interferon (IFN) produced by cancer antigen-specific cytotoxic T cells (CTL) and its downstream signaling pathway play an important role in cancer immunoediting process. Immune checkpoint blockade (ICB) such as anti-PD-1 antibody is an emerging cancer immunotherapy with high efficacy in the clinic, however, the clinical response has clear limitation. At present, many pre-clinical and clinical studies have been conducted to test the effective combination therapy with ICB that may enhance the anti-tumor immunity. Some type of Kampo medicines have long been expected to exert immunostimulatory effects as a biological response modifier (BRM), and in particular, Juzentaihoto has been known to modulate anti-immunity in various pre-clinical mouse cancer models. In this talk, I will present our recent data of cancer cell-host immune interaction in vivo by monitoring the antigen-specific CTL responses using bioluminescence imaging guide to dissect the immunological features of CTL during cancer immune editing process. I will also share our effort to test the combination of Juzentaihoto with anti-PD-1 immunotherapy in pre-clinical mouse cancer model.

600 Microarray patch-based adenovirus vaccines enable effective skin immunization

Recombinant live adenovirus (Ad) vectors represent a readily modular vaccine construct that can be engineered to include any antigen of interest, thereby enabling rapid vaccine development against a myriad of infectious pathogens, including coronaviruses. However, current approaches used for delivery, storage, and distribution of Ad vaccines hinder their full potential for effective global immunization campaigns. Here, we developed simple, effective, practical, and needle-free Ad vaccines based on microarray patches (MAPs) to enable sustainable mass vaccination against SARS-CoV-2. Rational formulation of live Ads with nonreducing sugars and mechanically strong carbohydrates into dissolvable MAPs enabled effective skin-targeted delivery of Ads and efficient cutaneous transduction as determined by in vivo live imaging of mice following skin application of MAPs integrating Ad vectors with a reporter gene. Immunogenicity assessment of dissolving MAP-based Ad vectors encoding SARS-CoV-2 proteins in mice demonstrated that skin immunization via MAP delivery of Ad-vectored COVID-19 vaccines elicited robust antigen-specific antibody responses, and these antibodies led to virus-specific neutralization activities, which were enhanced compared to those obtained with traditional intramuscular immunization. Furthermore, MAP delivery of Ad-vectored vaccines elicited potent cell-mediated immune responses, including polyfunctional virus-specific CD8+ and CD4+ T-cell responses in spleens and lungs of immunized mice, as determined by intracellular cytokine staining and flow cytometry, as well as antigen-specific cytotoxic T-cell responses in spleens of mice, as determined by lytic assay. Collectively, our results suggest that dissolvable MAPs could enable the development of skin-targeted Ad-based vaccines that may increase the effectiveness of global immunization programs for SARS-CoV-2 and other existing or future pathogens.

A Novel PD-L1-Containing MSLN Targeting Vaccine for Lung Cancer Immunotherapy.

Therapeutic tumor vaccines have become an important breakthrough in the treatment of various solid tumors including lung cancer. Dendritic cells (DCs)-based tumor vaccines targeting tumor-associated antigens (TAAs) play a key role in immunotherapy and immunoprevention. However, the weak immunogenicity of TAAs and low immune response rates are a major challenge faced in the application of therapeutic tumor vaccines. Here, we tested whether targeting an attractive target Mesothelin (MSLN) and PD-L1 immune checkpoint molecule to DCs in vivo would elicit therapeutic antitumor cytotoxic T lymphocyte (CTL) response. We generated specific MSLN fragment combined with PD-L1 and GM-CSF peptide immunogen (MSLN-PDL1-GMCSF) based on the novel anti-PD-L1 vaccination strategy we recently developed for the cancer treatment and prevention. We found that DCs loaded with MSLN-PDL1-GMCSF vaccine elicited much stronger endogenous anti-PD-L1 antibody and T cell responses in immunized mice and that antigen specific CTLs had cytolytic activities against tumor cells expressing both MSLN and PD-L1. We demonstrated that vaccination with MSLN-PDL1-GMCSF potently inhibited the tumor growth of MSLN+ and PD-L1+ lung cancer cells, exhibiting a significant therapeutic anti-tumor potential. Furthermore, PD-1 blockade further improved the synergistic antitumor therapeutic efficacy of MSLN-PDL1-GMCSF vaccine in immunized mice. In summary, our data demonstrated for the first time that this PD-L1-containing MSLN therapeutic vaccine can induce persistent anti-PD-L1 antibody and CTL responses, providing an effective immunotherapeutic strategy for lung cancer immunotherapy by combining MSLN-PDL1-GMCSF vaccine and PD-1 blockade.

Abstract 6347: Immunotherapeutic application of induced pluripotent stem cell technology: Rejuvenated BCMA-specific CD8+T cells for multiple myeloma

Abstract T cell regenerative medicine represents an immunotherapeutic approach using antigen-specific induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8+ cytotoxic T lymphocytes (CTL). Here, we report an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) to overcome exhaustion of antigen-specific CTL and revitalize them to fully functional antigen-specific memory T cells to target multiple myeloma (MM). First, we established the iPSC by reprogramming IFN-γ producing heteroclitic BCMA72-80 (YLMFLLRKI) peptide-specific CD8+ CTL via Sendai virus transduction of transcription factors, OCT3/4, SOX2, KLF4 and c-MYC. The BCMA-specific iPSC demonstrated high pluripotency potential and ability to differentiate into three key germ layers, as evidenced by expression of stem cell markers (SSEA-4, TRA1-60), germ differentiation markers (SOX-17 on Endoderm, Brachyury on Mesoderm, and Pax-6 on Ectoderm) and alkaline phosphatase. The polarization of iPSC was followed during embryoid body formation into mesoderm development, evidenced by activation of transcriptional regulators SNAI2, TBX3, PLVAP, HAND1 and CDX2. Furthermore, hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell development under feeder-free culture conditions in the presence of rectonectin. Upon differentiation, phenotypic characterization revealed fully mature T cells with high expression (> 95%) of CD3, CD45, TCRαβ and CD8αβ, which were predominantly CD45RO+ memory T cells with high activation (CD38) and costimulatory (CD28) molecule expression, while lacking immune checkpoints (CTLA4, PD1, LAG3, Tim3). This phenotype was aligned with their high proliferative (1,800-fold increase) capacity and effective anti-tumor cytotoxicity and Th1 cytokine (IFN-γ, IL-2, TNF-α) production against MM patients’ tumor cells in antigen-specific and HLA-A2-restricted manner. Their anti-MM activities were specifically directed against the parent heteroclitic BCMA72-80 peptide via a distinct sole T cell receptor clonotype. RNAseq analyses identified specific transcriptional pathways utilized by BCMA-specific HPC during their differentiation into CD8+ CTL, which include upregulation of transcriptional regulators determining CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, as well as downregulation of regulators controlling B and T cell interactions or CD4+ Th cells and inhibitory receptor development. In summary, these results highlight the processes and pathways mediating somatic T cell epigenetic reprogramming and differentiation into rejuvenated BCMA-specific CD8+ CTL with high proliferation and functional anti-MM activities, providing the framework for regenerative medicine as an adoptive immunotherapy to improve patient outcome in MM. Citation Format: Jooeun Bae, Shuichi Kitayama, Zach Herbert, Laurence Daheron, Nikhil Munshi, Shin Kaneko, Jerome Ritz, Kenneth Anderson. Immunotherapeutic application of induced pluripotent stem cell technology: Rejuvenated BCMA-specific CD8+T cells for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6347.

Blockade of TIM-3 and PD-1 enhances the antitumor effects of MAGE-A11 antigen-specific cytotoxic T lymphocytes

Cancer immunotherapy is an attractive approach for antigen-specific T cell-mediated antitumor therapy, especially for the induction of cytotoxic T lymphocytes (CTLs). An human leukocyte antigen (HLA)-A2-restricted melanoma-associated antigen-A11 (MAGE-A11) peptide was developed that exhibited a potent capacity to induce cytotoxicity towards MAGE-A11-positive breast cancer cells by activating CTLs. However, this antitumor immune response can be suppressed by inhibitory pathways. Programmed death-1 (PD-1) and T cell immunoglobulin domain and mucin domain 3 (TIM-3) pathways are two important pathways involved in the tumor-mediated immune suppression. The present study aimed to augment the efficacy of MAGE-A11 antigen-specific CTLs via blocking PD-1 and TIM-3. The results showed that the expression levels of PD-1 and TIM-3 were unregulated during T cell induction, expansion and target cell killing. Blockade of PD-1 and TIM-3 modulated T cell proliferation, transformation and survival. In addition, treatment of cells with antibodies against PD-1 and TIM-3 enhanced the cytotoxicity of MAGE-A11 antigen-specific CTLs against breast cancer cells. The aforementioned findings suggested that MAGE-A11 antigen-specific CTLs accompanied by PD-1 and TIM-3 blockade could be considered as a potential therapy approach for breast cancer.

Tumoral Expression of NAC1 Restrains Antitumor Immunity through the LDHA-mediated Immune Evasion

Abstract T cell-mediated antitumor immunity has an important role in cancer prevention and treatment; however, immunosuppressive tumor microenvironment (TME) constitutes a major contributor to immune evasion that weakens antitumor immunity. We report here that tumoral expression of nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) gene family, negatively impacts the CD8+ T cell - mediated antitumor immunity via through LDHA-mediated suppressive tumor microenvironment. We show that NAC1 could positively regulate the expression of lactate dehydrogenase A (LDHA) at transcriptional level, and LDHA-mediated lactic acid accumulation only inhibited cytokine production but also induced the exhaustion and apoptosis of CD8+ cytotoxic T lymphocytes (CTLs), impairing their cell-killing ability. We further show that in the immunocompetent and immunodeficient mice bearing melanoma, tumors with NAC1 depletion grew significantly slower and had elevated infiltration of tumor antigen specific CTLs following adoptive cell transfer, as compared with the control groups. These findings imply that tumor expression of NAC1 contributes substantially to immune evasion through its regulatory role in LDHA expression and lactic production. Thus, therapeutic targeting of NAC1 warrants further exploration as a potential strategy to reinforce cancer immunotherapy such as adoptive cell transfer of CTLs.

Induced pluripotent stem cell-derived rejuvenated cytotoxic T lymphocyte therapy for Epstein-Barr virus-associated lymphomas: application to clinical practice

Epstein-Barr virus (EBV)-associated lymphomas are common in Asia and exhibit a poor prognosis. As EBV antigens LMP1 and LMP2 are often expressed in EBV-associated lymphomas, these lymphomas should be a good target for antigen-specific cytotoxic T lymphocyte (CTL) therapy. However, CTLs continuously exposed to viral or tumor antigens often become exhausted. Antigen-specific CTLs generated from induced pluripotent stem cells are functionally rejuvenated, showing a strong antitumor effect on EBV-associated lymphomas and persistence in vivo. For feasible "off-the-shelf" therapy, we generated allogeneic EBV-specific CTLs in the cell processing center and prepared them for actual use in clinical settings.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Simple SummaryDeveloping new therapeutic cancer vaccines is of paramount importance to counteract tumor escape observed after conventional therapies in certain types of cancer. We have previously shown that the combination of two different vaccine platforms, targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. Here, we show that the heterologous prime-boost combination with a protein vaccine and a viral vector vesicular stomatitis virus immunologically reshapes the immune-excluded TC-1 tumor model as well as the inflamed MC-38 tumor model, leading to beneficial therapeutic efficacy. Furthermore, the treatment with a multi-epitope vaccine allowed us to appreciate the various repartition among three antigen-specific cytotoxic T-cell responses combined with the viral boost. The combination leads to improved efficacy in all animals and highlights the importance of combining tumor epitopes. Our vaccine strategy could be further extended to prophylactic cancer vaccines and beyond, for infectious diseases.Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.