Cytotoxic T Lymphocytes Therapy Research Articles

Prediction and identification of HLA-A*0201-restricted epitopes from cancer testis antigen CT23

ABSTRACT Cancer-testis antigen CT23 is a class of tumor-associated antigens (TAA) characterized by restricted expression in male germ cells and a variety of tumor tissues. Numerous studies have shown that CT23 is closely related to tumor cell viability, proliferation, metastasis and invasion. CT23 is immunogenic and can cause specific immune response in tumor patients. Therefore, it is considered to be one of the best target antigens for designing therapeutic tumor vaccines and T-cell-mediated tumor immunotherapy. In this study, we initially obtained seven HLA-A*0201-restricted CT23 epitope candidate peptides through the T cell epitope prediction program. Subsequently, a T2 cell binding assay revealed the potential binding of all candidate peptides with HLA-A2 molecules. Notably, peptide P7 (ALLVLCYSI) exhibited the highest affinity, as evidenced by a fluorescence index (FI) of 2.19. Dendritic cells (DCs) loaded with CT23 candidate peptide can stimulate CD8+T cell activation and proliferation, and compared with other candidate peptides, candidate peptide P7 is superior. The cytotoxic T lymphocytes (CTLs) stimulated by the peptide P7 had killing effect on tumor cells (HLA-A*0201+, CT23+), but no killing effect on tumor cells (HLA-A*0201−, CT23+). The CTLs induced by the peptide P7 also had a specific killing effect on T2 cells bearing the peptide P7. In summary, our findings suggest that the CT23 peptide P7 (ALLVLCYSI) can induce immune responses and holds potential for tumor-specific CTL therapy.

The safety and anti-tumor effect of multiple peptides-pulsed dendritic cells combined with induced specific cytotoxic T lymphocytes for patients with solid tumors

ObjectiveThe aim of this study was to explore the safety and efficacy of multiple peptide-pulsed autologous dendritic cells (DCs) combined with cytotoxic T lymphocytes (CTLs) in patients with cancer.MethodsFive patients diagnosed with cancer between November 2020 and June 2021 were enrolled and received DC-CTLs therapy. Peripheral blood was collected and antigenic peptides were analyzed. The phenotype and function of DC-CTLs and the immune status of patients were detected using flow cytometry or IFN-γ ELISPOT analysis.ResultsDCs acquired a mature phenotype and expressed high levels of CD80, CD86, CD83, and HLA-DR after co-culture with peptides, and the DC-CTLs also exhibited high levels of IFN-γ. Peripheral blood mononuclear cells from post-treatment patients showed a stronger immune response to peptides than those prior to treatment. Importantly, four of five patients maintained a favorable immune status, of which one patient’s disease-free survival lasted up to 28.2 months. No severe treatment-related adverse events were observed.ConclusionOur results show that multiple peptide-pulsed DCs combined with CTLs therapy has manageable safety and promising efficacy for cancer patients, which might provide a precise immunotherapeutic strategy for cancer.

Prevention and management of human cytomegalovirus in pediatric HSCT recipients: A review.

Cytomegalovirus (CMV), like other herpesviruses, has the unique ability to establish latent infection with subsequent reactivation during periods of stress and immunosuppression. Herpesviruses cause potentially devastating disease, particularly in hematopoietic stem cell transplant (HSCT) recipients. CMV is especially of concern in HSCT recipients given the high community seroprevalence, high risk of reactivation and high risk of transmission from HSCT donors to recipients causing primary infection after transplantation. The risk of CMV infection and severity of CMV disease varies depending on the underlying disease of the HSCT recipient, donor and recipient CMV status prior to HSCT, type of conditioning therapy in preparation for HSCT, allogeneic versus autologous HSCT, donor graft source, timing of infection in relation to HSCT, and other patient comorbidities. Different strategies exist for prevention (e.g., preemptive therapy vs. universal prophylaxis) as well as management of CMV disease (e.g., antiviral therapy, augmenting immune reconstitution, cytotoxic T-cell therapy). The purpose of this narrative review is to discuss diagnosis, prevention, and management of CMV infection and disease at different stages of HSCT, including key points illustrated through presentations of complex cases and difficult clinical scenarios. Traditional and novel strategies for CMV management will be discussed in the context of these unique clinical cases.

Research advances in the targeted therapy and immunotherapy of Wilms tumor: a narrative review.

Wilms tumor is the most common pediatric abdominal solid tumor, and its treatment has been a focus of research. For now, the 5-year survival rate of children with Wilms tumor is about 90%. It is difficult to make further progress simply by the improvement of the existing treatments (multi-modal therapy). Therefore, targeted therapy and immunotherapy which have high accuracy and few side effects began to be considered for the treatment of Wilms tumor. At present, though targeted therapy and immunotherapy are rarely used in the treatment of Wilms tumor except in clinical trials, there are dozens of clinical trials research them around the world. The sites in targeted therapy research are mainly focused on insulin-like growth factor 2 (IGF2) pathway, anti-angiogenesis, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, and some miRNAs, etc. And there are three types of study in Wilms tumor immunotherapy, which are inhibition of the COX-2 pathway, chimeric antigen receptor (CAR)-T cell therapy, and multi-tumor associated antigen (TAA)-specific cytotoxic T lymphocytes (CTL) therapy. Among them, the phase I clinical trial of multi-TAA-specific CTL (MTAA-CTL) therapy has been completed, and the results are very satisfactory. In this narrative review, we review the basic research and relevant clinical research on targeted therapy and immunotherapy for Wilms tumor.

TCR repertoire characteristics predict clinical response to adoptive CTL therapy against nasopharyngeal carcinoma

ABSTRACT The past decade has witnessed the gradual and steady progress of adoptive T cell therapy in treating various types of cancer. In combination with gemcitabine and carboplatin chemotherapy, we previously conducted a clinical trial, NCT00690872, to treat Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) patients with autologous EBV-expanded cytotoxic T lymphocytes (CTLs). While achieving a 2-year overall survival rate of 62.9%, this trial failed to induce an anti-tumor response in a sizable fraction of patients. Thus, the identification of benchmarks capable of evaluating CTL products and predicting clinical immunotherapeutic efficacy remains an urgent need. We conducted T cell receptor (TCR) repertoire sequencing to assess EBV-expanded infusion-ready CTL products. To depict the overall repertoire landscape, we evaluated the individual repertoire diversity by Shannon entropy, and, compared the inter-patient CDR3 similarity to estimate T cells expanded by common antigens. With a recently developed bioinformatics algorithm, termed Motif Analysis, we made a machine-learning prediction of structural regions within the CDR3 of TCRβ that associate with CTL therapy prognosis. We found that long term survivors, defined as patients surviving longer than two years, had a higher CTL repertoire diversity with reduced inter-patient similarity. Furthermore, TCR Motif Analysis identified 11 structural motifs distinguishing long term survivors from short term survivors. Specifically, two motifs with a high area under the curve (AUC) values were identified as potential predictive benchmarks for efficacious CTL production. Together, these results reveal that the presence of diverse TCR sequences containing a common core motif set is associated with a favorable response to CTL immunotherapy against EBV-positive NPC.

Atypical malignancies after intestinal transplantation in children: A European single-centre experience.

Post-transplant malignancies, that is, lymphomas, are a recognized complication in intestinal transplant recipients but are mostly secondary to EBV infection. There is an increased risk for malignancies in unusual sites in intestinal transplant recipients as compared to other solid organ transplants and the general population. To evaluate the incidence, course, and outcome of unusual malignancies in children after ITx. Retrospective analysis of children who underwent ITx for primary digestive disorders at Birmingham Children's Hospital between January 1989 and December 2017. Ninety-eight intestinal transplants were performed in 90 children (49 males and 41 females) with an underlying primary digestive disorder. Median age was 2.7years (0.6-16.2), and median weight was 14.5kg (5.7-53.2) at the time of transplant. Within this cohort, we identified four cases of unusual malignancies at rare sites of presentation. One patient developed cerebral PTLD, two patients were diagnosed with SMT, located at the stomal orifice and in cervicothoracic paravertebral area, respectively, and the last patient developed a retroperitoneal angiosarcoma. Unfortunately, the overall patient outcome was poor in all but one child with SMT, who currently survives with cytotoxic T-cell therapy. Unusual malignancies can occur in approximately 5% of children following ITx. A high index of suspicion is required for a timely diagnosis and adequate treatment.

The efficacy and safety of Epstein-Barr virus-specific antigen peptide-activated cytotoxic T-cells treatment for refractory or recurrent angioimmunoblastic T-cell lymphoma: A prospective clinical observational study.

The efficacy and safety of Epstein-Barr virus (EBV)-specific antigen peptide-activated cytotoxic T lymphocytes (CTLs) in the treatment of refractory or recurrent angioimmunoblastic T-cell lymphoma (AITL) was determined in this prospective one-arm clinical study. Seven males and two females were enrolled with a median age of 70 years. The tumor stages were all stage III and IV. All patients had group B symptoms and IPI scores of 3 to 5 points. All patients received chemotherapy before CTLs infusion which the median chemotherapy cycle was three. The diseases states before CTLs included five cases of disease progression (PD), two cases of recurrence (R), and two cases with residual lesions after chemotherapy. Eight patients received HLA-haploidentical EBV-specific CTLs, and one patient chose autologous CTLs. The number of transfused cells was 1.67 to 2.38 × 1010 for one course of CTLs therapy. One patient was treated with three courses of CTLs, three patients were treated with two courses of CTLs, and five patients were treated with one course of CTLs. During the infusion, eight patients had fever, one patient had rash, and no graft-vs-host diseases were observed. The EBV-DNA decreased by more than two orders of magnitude in six patients, and the response rate was 66.7%. Two patients of PD status achieved complete remission (CR), one patient of PD status achieved partial remission, two patients with residual lesions after chemotherapy achieved CR, and four patients had no response. The objective remission rate was 55.6%. After the median follow-up of 14.5 months, five patients died, and three patients were completely relieved while one patient was lost during follow-up. The 3-year overall survival was 44.4% and 3-year progression-free survival was 33.3%. EBV-specific antigen peptide-activated CTLs showed positive effect in certain patients with refractory and recurrent AITL with high clinical safety.

Resistant or refractory cytomegalovirus infections after hematopoietic cell transplantation: diagnosis and management.

Refractory or resistant cytomegalovirus (CMV) infections are challenging complications after hematopoietic cell transplantation (HCT). Most refractory or resistant CMV infections are associated with poor outcomes and increased mortality. Prompt recognition of resistant or refractory CMV infections, understanding the resistance pathways, and the treatment options in HCT recipients are imperative. New definitions for refractory and resistant CMV infections in HCT recipients have been introduced for future clinical trials. Interestingly, refractory CMV infections are more commonly encountered in HCT recipients when compared with resistant CMV infections. CMV terminase complex mutations in UL56, UL89, and UL51 could be associated with letermovir resistance; specific mutations in UL56 are the most commonly encountered in clinical practice. Finally, brincidofovir, maribavir, letermovir, and CMV-specific cytotoxic T-cell therapy expanded our treatment options for refractory or resistant CMV infections. Many advances have been made to optimize future clinical trials for management of refractory or resistant CMV infections, and to better understand new resistance mechanisms to novel drugs. New drugs or strategies with limited toxicities are needed to improve outcomes of difficult to treat CMV infections in HCT recipients.

Third-Party BK Virus Specific Cytotoxic T Lymphocyte Therapy for Hemorrhagic Cystitis Following Allotransplantation

Background. BK virus associated hemorrhagic cystitis (BKV-HC) is a major complication of allogeneic hematopoietic stem cell transplantation (AHSCT), [Bedi, A., et al. JCO 1995] particularly in recipients of alternative donor transplants, which are being performed with increasing frequency [O'Donnell, P.H., et al, BBMT 2009]. BKV-HC typically results in painful hematuria, urinary obstruction, and renal dysfunction, with few effective therapeutic options [Seber, A., et al., BMT 1999]. Methods. We assessed the feasibility, safety, and efficacy of administering most closely human leukocyte antigen (HLA)-matched third party BKV-specific cytotoxic T lymphocytes (CTLs), generated from 26 healthy donors and banked for off-the-shelf use. The cells were infused into 47 patients who developed BKV-HC following AHSCT. Comprehensive clinical assessments and correlative studies were performed. We compared their toxicity and survival to a control group of 85 AHSCT patients with BKV-HC treated with standard of care, using inverse probability of treatment weighted (IPTW) estimation to correct for bias due to non-randomization. The control group was matched for age, gender, graft versus host disease prophylaxis, conditioning regimen, AHSCT graft source, and both initial and maximum grade of BKV-HC. Results. On average, BKV disease status improved from 7 to 90 days post CTL infusion, with the primary response endpoint scored on day 28. Among 31 evaluable patients on day 28 who received a single BKV CTL infusion, 18 had a complete response (CR) and 9 a partial response (PR), for an overall response rate of 87%, which increased to 100% among evaluable patients at day 90 (Figure 1). Four of 7 patients who received multiple (1-5) CTL infusions for persistent disease following the first infusion are alive without BKV-HC. Median survival from the date of transplant in the BKV CTL group was 772 days, compared to a median of 171 days for the controls (IPTW adjusted log rank test p-value = 0.02) as shown in Figure 2. One BKV CTL patient with a history of skin graft versus host disease (GVHD) developed grade 3 gastrointestinal GVHD 64 days post CTL infusion, which resolved completely with systemic steroids. There were no cases of graft failure or infusion-related toxicities. BKV CTLs were identified in patient blood samples up to one month post-infusion. Conclusion. This is the largest series of AHSCT patients treated to date with BKV CTLs. The high response rate we observed in this study appeared to confer a survival benefit compared to a historically controlled cohort with BKV-HC treated with standard of care alone. This study demonstrated that administering third party, off-the-shelf banked BKV CTLs is feasible and safe. The safety profile, high response rate, and logistical ease with which potent off-the-shelf BKV CTL therapy were generated and administered warrants further investigation for patients with BKV-HC who have limited alternative therapeutic options. Disclosures Chemaly: ADMA Biologics: Other: Personal fees; Gilead: Research Funding; Janssen: Other: Personal fees; Ansun: Other: Personal fees; Oxford Immunotec: Research Funding; Chimerix: Research Funding; Ablynx: Other: Personal fees; Merck: Research Funding; Clinigen: Other: Personal fees; Gilead: Other: Personal fees; Ansun Pharmaceuticals: Research Funding; Chimerix: Other: Personal fees; Oxford Immunotec: Other: Personal fees; Merck: Other: Personal fees; ReViral: Other: Personal fees. Ciurea:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding. Kebriaei:Kite: Honoraria; Jazz: Consultancy; Pfizer: Honoraria; Amgen: Research Funding. Oran:Astex pharmaceuticals: Research Funding; AROG pharmaceuticals: Research Funding. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Research Highlights

Research Highlights

PNEUMONITIS SECONDARY TO EBV-SPECIFIC CYTOTOXIC T-LYMPHOCYTES

SESSION TITLE: Diffuse Lung Disease 1 SESSION TYPE: Fellow Case Reports PRESENTED ON: 10/07/2018 03:30 pm - 04:30 pm INTRODUCTION: Novel immune based therapies are now being used for a wide range of malignancies including post-transplantation lymphoproliferative disorders (PTLD), however, the short and long term risk of these therapies remains unclear. Here, we present a case of a patient who developed hypoxemia one day after receiving an infusion of EBV-specific cytotoxic T-lymphocytes. CASE PRESENTATION: A 66-year-old male presented to the hospital with 4 days of progressive dyspnea, cough, and hypoxemia. He had a history of end stage renal disease and EBV-positive cadaveric renal transplantation which was complicated by EBV viremia and PTLD. Two months prior, the patient began therapy with HLA-matched EBV-specific T-lymphocytes after failing several immunosuppressive and chemotherapeutic regimens. His most recent infusion was 1 day prior to symptom onset. On admission, oxygen saturation was 91% on room air and 96% on 2 liters of oxygen by nasal cannula. Physical exam at that time was notable for faint crackles throughout all lung fields. Chest radiograph was negative for infiltrates or effusions although computed tomography imaging of the chest revealed diffuse bilateral ground glass opacities and patchy airspace disease with an upper lobe predominance (see Figure 1). Bronchoscopy with transbronchial biopsy revealed interstitial inflammation and fibrosis, hyperplasia of type II pneumocytes, and scattered foci of organizing pneumonia consistent with active pneumonitis. Bronchoalveolar lavage demonstrated a lymphocyte predominant cell distribution with 56% lymphocytes, 41% macrophages, and 3% neutrophils. BAL and biopsy were negative for viral, fungal, and bacterial elements. With supportive care alone, the patient improved over the course of 5 days and was discharged to a short term rehabilitation facility. DISCUSSION: PTLD is a serious complication for patients undergoing organ transplantation. EBV-specific cytotoxic T-cells have shown promising results in the treatment of some of these patients, however, emerging evidence suggests that pulmonary complications are a consequence of this therapy. Indeed, in a series of 39 patients there was 1 reported death due to pneumonitis. Moreover, outcomes in another study of 114 patients found that 1 patient developed severe pulmonary infiltrates within one week of receiving EBV-specific T-cells. This patient did not suffer long-term sequelae from this complication. Our case suggests that pneumonitis from EBV-specific T-lymphocytes is characterized by a histopathological pattern of organizing pneumonia and a lymphocyte predominant cellular distribution. CONCLUSIONS: EBV-specific cytotoxic T-cell therapy causes acute pneumonitis in some patients. Additional studies will be important to understand the true incidence of pneumonitis after EBV-specific therapies, and for determining the factors that predispose or prevent the development of this therapeutic complication. Reference #1: Heslop, H. E., Slobod, K. S., Pule, M. A., et al. Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood. 2010; 115(5):925-935. Reference #2: Rooney, C. M., Smith, C. A., Ng, C. Y., et al. Infusion of Cytotoxic T Cells for the Prevention and Treatment of Epstein-Barr Virus–Induced Lymphoma in Allogeneic Transplant Recipients. Blood. 1998; 92(5):1549-1555. Reference #3: Dierickx, D., and Habermann, T.M. Post-Transplantation Lymphoproliferative Disorders in Adults. N Engl J Med. 2018; 378:549-562. DISCLOSURES: No relevant relationships by Robert Hilton, source=Web Response No relevant relationships by Carl Nieweld, source=Web Response No relevant relationships by ross summer, source=Web Response

PD-1 blockade potentially enhances adoptive cytotoxic T cell potency in a human acute myeloid leukaemia animal model

ABSTRACTObjectives: Acute myeloid leukaemia (AML) is a malignant haematological disease that remains difficult to cure. Cytotoxic T cell (CTL) adoptive infusion therapy may be conducive to tumour remission by boosting physical immunity. Furthermore, programmed death receptor-1 (PD-1) blockade immunotherapy has shown tremendous success in many cancer therapies.Method: We attempted to combine these two immunotherapy strategies to intervene in AML by generating AML cellspecific cytotoxic T lymphocytes in vitro and in vivo with an AML cell strain expressing specific antigens.Results: First, we observed that peripheral blood mononuclear cells (PBMCs) could be induced to generate large numbers of CD8+ CTL cells through immune stimulation. In addition, these CD8+ cells could effectively recognize a human AML cell line and exert cytotoxicity. In animal tests, PD-1 blockade combined with CTL infusion could induce significantly more AML tumour reduction than either treatment alone. This synergistic effect was thought to be connected to immune modulation enhancement, as regulatory T cells (Tregs) in the peripheral blood (PB) were found to be suppressed.Conclusions: This finding suggested the potential application of PD-1 blockade in AML. The present work demonstrated an excellent synergistic tumour therapeutic effect of PD-1 blockade and CTL therapy compared with either treatment alone.

IPSC-derived rejuvenated T-cell therapy for Epstein-Barr virus-associated lymphomas

Extranodal natural killer (NK) /T-cell lymphoma (ENKL), nasal-type, is an aggressive lymphoma that is relatively common in Asia and is resistant to anthracycline-based chemotherapy. Antigen-specific cytotoxic T-lymphocyte (CTL) therapy can induce durable remission in selected tumors, such as melanomas and virus-related tumors. ENKLs are commonly infected with the Epstein-Barr virus (EBV) ; therefore, CTL therapy can be effective for these lymphomas. However, CTLs that are continuously exposed to viral or tumor antigens usually get exhausted. Antigen-specific CTLs generated from iPSCs have a higher proliferative capacity and longer telomeres than original CTLs and are functionally rejuvenated (rejuvenated T cell, rejT). For clinical translation, the tumorigenic potential of iPSCs and the malignant transformation of differentiated iPSCs are major safety concerns. To address these issues, we introduced inducible caspase-9 (iC9) -based safeguard system into iPSCs. iC9-rejTs exert strong anti-tumor effects against EBV-infected tumors in vivo, and the iC9 suicide gene system provides a reliable safeguard for rejT therapy. We initiated a preclinical study using this novel immunotherapy for EBV-associated lymphoma. We believe that rejT therapy can be a potential safe approach as an "off-the-shelf" therapy for refractory ENKL in the future.

Delivery of foreign cytotoxic T lymphocyte epitopes to tumor tissues for effective antitumor immunotherapy against pre-established solid tumors in mice.

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants). In addition, tumor cells can downregulate the levels of major histocompatibility complex class I (MHC-I) molecules on cell surface due to immune pressure. As a result, some tumor cells can escape the immune pressure bestowed by CTLs, resulting in treatment failure. To address these difficulties, a new approach is developed to deliver foreign high-affinity CTL epitopes to tumor tissues utilizing pH-responsive "smart" microparticles (MPs). These MPs could encapsulate CTL peptide epitope, release the peptide under acidic condition encountered in tumor tissues and enhance CTL activation. Mice bearing pre-established tumor as "antigen-loss variant" solid tumor models were administered intratumorally with MPs containing the CTL peptide, which showed 100% survival following the treatment. In contrast, all control mice died from tumor. Significant protection from tumor-induced death was also observed with systemic administration of CTL peptide-MPs. The therapeutic efficacy can be attributed to enhanced delivery of the epitope to tumor tissues, presentation of the epitope by tumor cells as well as tumor stromal cells and/or generation of epitope-specific CTLs by the peptide-containing MPs. These findings offer a promising new direction for treating established solid tumor using CTL therapy.

Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines.

Epstein-Barr virus-related post-transplant lymphoproliferative disorders are recognized as a significant cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. To better define current understanding of post-transplant lymphoproliferative disorders in stem cell transplant patients, and to improve its diagnosis and management, a working group of the Sixth European Conference on Infections in Leukemia 2015 reviewed the literature, graded the available quality of evidence, and developed evidence-based recommendations for diagnosis, prevention, prophylaxis and therapy of post-transplant lymphoproliferative disorders exclusively in the stem cell transplant setting. The key elements in diagnosis include non-invasive and invasive methods. The former are based on quantitative viral load measurement and imaging with positron emission tomography; the latter with tissue biopsy for histopathology and detection of Epstein-Barr virus. The diagnosis of post-transplant lymphoproliferative disorder can be established on a proven or probable level. Therapeutic strategies include prophylaxis, preemptive therapy and targeted therapy. Rituximab, reduction of immunosuppression and Epstein-Barr virus-specific cytotoxic T-cell therapy are recommended as first-line therapy, whilst unselected donor lymphocyte infusions or chemotherapy are options as second-line therapy; other methods including antiviral drugs are discouraged.

PD-L1 on Antigen-Presenting Cells Facilitate the Induction of Antigen-Specific Cytotoxic T Lymphocytes

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, it has been shown that engagement of PD-1 on T cells by PD-L1 on tumor cells is associated with the immune escape of tumors, and recent clinical trials have highlighted the anti-tumor efficacy of blockade of the PD-1/PD-L1 pathway. Along with costimulatory molecules such as CD80 and CD86, PD-L1 is also highly expressed on dendritic cells (DCs), which are professional antigen-presenting cells (APCs) that are widely used to generate antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy. However, the roles of PD-L1 on APCs have not been well examined. Thus, we evaluated the roles of PD-L1 on APCs in the induction of cytomegalovirus (CMV)-specific CTLs (CMV-CTLs).First, peripheral blood mononuclear cells (PBMCs) were obtained from five CMV-seropositive healthy donors (HLA-A*02:01, n = 3; A*24:02, n = 1; B*07:02, n = 1). Mature DCs were generated from adherent PBMCs by stimulation with GM-CSF/IL-4 and maturational cytokines (TNF-a, IL-1b, IL-6, and PGE2), and freshly isolated CD3+ T cells were stimulated with mature DCs pulsed with CMV pp65-derived HLA-restricted peptides (NLVPMVATV for HLA-A*02:01, QYDPVAALF for A*24:02, and TPRVTGGGAM for B*07:02) in the presence or absence of anti-PD-L1 blocking antibody. After 14 days of culture, the presence of anti-PD-L1 antibody resulted in a less efficient induction of CMV-CTLs (Figure 1), suggesting that PD-L1 might play a positive role in the induction of antigen-specific CTLs. For further evaluation, we generated K562-based artificial APCs (aAPCs), which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1 (named K562, K562+CD80/86, K562+PD-L1, and K562+CD80/86+PD-L1). CD3+ T cells isolated from nine CMV-seropositive healthy donors (HLA-A*02:01, n = 4; A*24:02, n = 4; B*07:02, n = 1) were stimulated weekly for 28 days using HLA-restricted CMV peptide-pulsed K562-based aAPCs. K562+CD80/86+PD-L1 led to significantly higher induction of CMV-CTLs than K562 or K562+CD80/86 (Figure 2). Since the original K562 cells slightly express CD80, we completely knocked out the CD80 expression in the cells using the CRISPR/Cas9 system, and then we transduced the cells with or without PD-L1 (named CD80KO K562+PD-L1 and CD80KO K562, respectively) to determine whether PD-L1 itself has a stimulatory effect on the induction of CMV-CTLs. CD80KO K562 and CD80KO K562+PD-L1 induced the same amount of CMV-CTLs, suggesting that PD-L1 itself does not have a stimulatory effect for the induction of CMV-CTLs. Phenotypic analysis showed that CMV-CTLs induced by K562+CD80/86+PD-L1 contained more CD45RA- CD62L+ central memory T cells, which persist longer and are more effective in vivo after adoptive transfer in CTL therapy, than CMV-CTLs induced by other K562-based aAPCs. In addition, when compared with CMV-CTLs induced by other K562-based aAPCs, CMV-CTLs induced by K562+CD80/86+PD-L1 did not express higher levels of exhaustion markers such as PD-1 and TIM-3, and exhibited similar levels of IFN-gamma and IL-2 production and CD107a expression in response to the specific peptide stimulation. Furthermore, to evaluate the effect of K562-based aAPCs on the induction of tumor antigen-specific CTLs, CD3+ T cells were isolated from three HLA-A*24:02-positive healthy donors and stimulated with HLA-A*24:02-restricted WT1 peptide-pulsed K562-based aAPCs weekly. After four stimulations, only K562+CD80/86+PD-L1 could clearly expand the WT1-specific CTLs from all of the three donors (Figure 3).In conclusion, our findings demonstrate that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of antigen-specific CTLs without causing excessive differentiation or functional exhaustion of the induced CTLs. These results suggest that stimulation with K562+CD80/86 might result in the activation-induced cell death of antigen-specific CTLs and that PD-L1 might be involved in fine-tuning excessive stimulation of CD80/86. Further analyses to determine the mechanisms of our findings are warranted. Our results also highlight the possibility that K562+CD80/86+PD-L1 has therapeutic potential as novel aAPCs for the generation of CTLs for adoptive immunotherapy. [Display omitted] DisclosuresKiyoi:Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Presentation of available CTL epitopes that induction of cell-mediated immune response against HIV-1 Koran clade B strain using computational technology.

Theoretical predicting cytotoxic T lymphocyte (CTL) epitopes are an important tool in vaccine design and CTL therapy for enhancing our understanding of the cellular immune system. We would like to identify available CTL epitopes against HIV-1 Korean clade B. CTL activity was assessed in freshly isolated peripheral blood mononuclear cells from Korean HIV patients in order to assess whether these CTL epitopes induce a cell-mediated immune response (CMI). NetCTLpan1.1 software, which is the most popular prediction computer software package, and full atom-based simulation (FABS), which is a 3D modelling system for binding activity between epitopes and human leucocyte antigen (HLA) molecules, were used to predict the peptide-spanning Env region binding to HLA-A*24:02, HLA-A*02:01 and HLA-B*15:01, which are frequently found in the Korean population. Granzyme B and interferon-γ ELISPOT assays were used to determine whether identified CTL epitopes induce CMI. Three HIV-1 Korean clade B-specific Env CTL epitopes were identified: Gp41-RYL and Gp41-RQG are localized within gp41, and Gp120-LLQ within gp120. In in vitro assays using granzyme B ELISPOT, Gp120-LLQ and Gp41-RQG induced epitope-specific CTL responses in HLA-restricted cells. In ex vivo assay using IFN-γ ELISPOT, cell-mediated immune responses to Gp41-RYL were present in 50% of HLA-matched patients, and responses to Gp120-LLQ and Gp41-RQG were found in 33% of HLA-matched patients. In this study, we found that a prediction pipeline for CTL epitopes might be based on the most popular computer prediction software and FABS methods. Our results suggest that these CTL epitopes may provide useful tools and information for the development of a therapeutic vaccine against HIV-1 Korean clade B.

Post-transplant lymphoproliferative disease and other malignancies after pediatric cardiac transplantation: an evolving landscape.

Post-transplant lymphoproliferative disorders (PTLDs) remain a significant cause of morbidity and mortality after pediatric solid organ transplantation (SOT). PTLD treatment outcomes have improved steadily over the past decade, in large part due to an enhanced understanding of the disease process, newer immunosuppression regimens, and implementation of evolving chemotherapeutic treatment protocols. New therapies continue to be employed to treat PTLDs while maintaining normal allograft function in SOT recipients. These include use of immunosuppressant medications with antitumor activity (mammalian target of rapamycin inhibitors), monoclonal antibody therapies, and the advent of cytotoxic T-cell therapy. Treatment methods to render latent Epstein-Barr virus (EBV)-infected tumor cells more susceptible to antiviral agents continue to be investigated. PTLD remains a significant potential complication after SOT, particularly in pediatric patients who are more likely to be EBV-negative at the time of transplant and subsequently undergo EBV seroconversion. Risk for PTLD may be reduced by employing strategies such as EBV prophylaxis in seronegative patients, minimizing overall intensity of immunosuppression, and utilizing newer agents that have both immunosuppressive and antiproliferative properties. Treatment outcomes for PTLD have steadily improved over the past decade, related in part to the availability of monoclonal antibody therapies and refined chemotherapeutic regimens.

The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) can result in robust and durable antitumor responses. Tumor-infiltrating CTLs produce IFNγ and mediate antitumor activity, but they simultaneously induce counter-regulatory immunosuppressive mechanisms in the tumor by recruiting monocytic myeloid-derived suppressor cells (MDSCs) that limit their proliferation and effector function. Using a murine model of adoptive immunotherapy for B16 melanoma, we developed a strategy to augment CTL activity by downregulating immunosuppression by MDSCs. Intravenous injection of transgenic pmel-1 CTLs into tumor-bearing mice, resulted in their infiltration into the tumor, but this was accompanied by the accumulation of large numbers of monocytic MDSCs (M-MDSCs). These cells hampered CTL function and reduced their numbers in the tumor. We determined that one mechanism responsible for this immunosuppression was the production of nitric oxide (NO) by MDSCs in the tumor. Therefore, mice were given the NO scavenger carboxy-PTIO (C-PTIO) on the day after CTL transfer. This led to the restoration of impaired proliferative capacity and function of the CTLs, resulting in sustained suppression of tumor growth. Thus, we conclude that CTL therapy can be improved by counter-acting immunosuppression. Targeting NO, one mediator of the immunosuppressive activity of M-MDSCs, may be an appropriate strategy to restore impaired CTL function and improve the efficacy of immunotherapy.

Activated cytotoxic T-lymphocyte immunotherapy is effective for advanced oral and maxillofacial cancers.

Conventional cancer treatments are surgery, radiotherapy, and chemotherapy, but treatment efficiency is insufficient and cancer recurrence is common. Immunotherapy has been added as an important cancer treatment component, but no reports on its efficacy in oral and maxillofacial cancers exist. We evaluated the clinical efficacy of adoptive immunotherapy using ex vivo-activated cytotoxic T lymphocytes (CTL) in the treatment of 7 patients with advanced oral and maxillofacial cancers with stage IV disease at diagnosis. The mean follow-up period was 26.2 months. Phenotype of the lymphocyte assay revealed that the percentage of CD4(+) T cells decreased and that of CD8(+) T cells increased among infused lymphocytes compared to that in unstimulated peripheral blood mononuclear cells (PBMCs), and infused lymphocytes produced a significantly higher level of IFN-γ than PBMCs or tumor cells alone. In a representative patient who refused surgery tumor regression was confirmed after CTL infusion. Computed tomography clearly indicated a significant reduction in tumor size followed by the complete disappearance of the tumor. Histological examination showed that the cancers in patients receiving CTL therapy were heavily infiltrated with lymphocytes. The other 2 patients who received CTL therapy as adjuvant therapy showed neither recurrent disease nor new disease lesions. The 1-year survival rates showing response and those with progressive disease were 100 and 25%, respectively. Moreover, no significant adverse reactions were reported during the study period. CTL therapy remains in the early stages of treatment options, but it has potential as a valuable treatment and improvement of quality of life for patients with otherwise incurable cancers.