Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome.

Mitochondrial DNA (mtDNA) leakage into the cytoplasm can occur when cells are exposed to noxious stimuli. Specific sensors recognize cytoplasmic mtDNA to promote cytokine production. Cytoplasmic mtDNA can also be secreted extracellularly, leading to sterile inflammation. However, the mode of secretion of mtDNA out of cells upon noxious stimuli and its relevance to human disease remain unclear. Here, we show that pyroptotic cells secrete mtDNA encapsulated within exosomes. Activation of caspase-1 leads to mtDNA leakage from the mitochondria into the cytoplasm via gasdermin-D. Caspase-1 also induces intraluminal membrane vesicle formation, allowing for cellular mtDNA to be taken up and secreted as exosomes. Encapsulation of mtDNA within exosomes promotes a strong inflammatory response that is ameliorated upon exosome biosynthesis inhibition in vivo. We further show that monocytes derived from patients with Behçet's syndrome (BS), a chronic systemic inflammatory disorder, show enhanced caspase-1 activation, leading to exosome-mediated mtDNA secretion and similar inflammation pathology as seen in BS patients. Collectively, our findings support that mtDNA-containing exosomes promote inflammation, providing new insights into the propagation and exacerbation of inflammation in human inflammatory diseases.

Bayesian predictive model averaging approach to joint longitudinal-survival modeling: Application to an immuno-oncology clinical trial.

In immuno-oncology clinical trials, multiple immunological biomarkers are usually examined over time to comprehensively and appropriately evaluate the efficacy of treatments. Because predicting patients' future survival statuses on the basis of such recorded longitudinal information might be of great interest, joint modeling of longitudinal and time-to-event data has been intensively discussed as a toolkit to implement such a prediction. To achieve a desirable predictive performance, averaging over multiple candidate predictive models to account for the model uncertainty might be a more suitable statistical approach than selecting the single best model. Although Bayesian model averaging can be one of the approaches, several problems related to model weights with marginal likelihoods have been discussed. To address these problems, we here propose a Bayesian predictive model averaging (BPMA) method that uses Bayesian leave-one-out cross-validation predictive densities to account for the subject-specific and time-dependent nature of the prediction. We examine the operating characteristics of the proposed BPMA method in terms of the predictive accuracy (ie, the calibration and discrimination abilities) in extensive simulation studies. In addition, we discuss the strengths and limitations of the proposed method by applying it to an immuno-oncology clinical trial in patients with advanced ovarian cancer.

Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.

WT1 Trio Peptide-Based Cancer Vaccine for Rare Cancers Expressing Shared Target WT1.

No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers.

Persistence of SARS-CoV-2 neutralizing antibodies and anti-Omicron IgG induced by BNT162b2 mRNA vaccine in patients with autoimmune inflammatory rheumatic disease: an explanatory study in Japan.

Background: Autoimmune inflammatory rheumatic disease (AIRD) patients are at high risk of the coronavirus disease 2019 (COVID-19), but the medium-term effects of immunosuppressants on vaccine efficacy are unknown. We investigated the duration of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and Omicron variant in AIRD patients administered with two doses of the BNT162b2 (Pfizer-BioNTech) vaccine. Methods: Serum-neutralizing antibody (NAb) and anti-receptor-binding domain (RBD)/spike antibody levels were measured. Short- and medium-term effects of immunosuppressants were analyzed pre-vaccination (Term 1) and 14-42 days (Term 2) and 100-200 days (Term 3) after the second vaccination. Findings: From Feb 1, 2021, to Feb 28, 2022, 439 AIRD patients and 146 healthy controls were investigated. The seropositivity rate and log10-NAb titers were significantly lower in AIRD patients than in controls at Terms 2 and 3. In rheumatoid arthritis patients, tumor necrosis factor-α inhibitors (TNFis) at Term 3, and older age, glucocorticoids, and abatacept at Terms 2 and 3 were risk factors for reduced responses. Anti-Omicron RBD/spike IgG levels strongly correlated with NAb titers. Interpretation: Glucocorticoids, TNFis, and abatacept treatments negatively affect the longevity of humoral responses to SARS-CoV-2, including Omicron, after two vaccine doses. These findings may inform the timing of additional vaccination for AIRD patients. Funding: Cloud Funding of Peace Winds Japan; Center of Innovation Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Japan Society for the Promotion of Science KAKENHI; Japan Agency for Medical Research and Development; Kansai Economic Federation; Mitsubishi Zaidan; and Research Grant from Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology.

The proliferative activity levels of each immune cell population evaluated by mass cytometry are linked to the clinical phenotypes of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, and many peripheral immune cell populations (ICPs) are thought to be altered according to the course of the disease. However, it is unclear which ICPs are associated with the clinical phenotypes of SLE. We analyzed peripheral blood mononuclear cells (PBMCs) of 28 SLE patients using mass cytometry and identified 30 ICPs. We determined the proliferative activity of ICPs by measuring the proportion of cells expressing specific markers and Ki-67 among CD45+ cells (Ki-67+ proportion). We observed an increased Ki-67+ proportion for many ICPs of SLE patients and examined the association between their Ki-67+ proportions and clinical findings. The Ki-67+ proportions of five ICPs [classical monocyte (cMo), effector memory CD8+ T cell (CD8Tem), CXCR5- naive B cell (CXCR5- nB), and CXCR5- IgD-CD27- B cell (CXCR5- DNB)] were identified as clinically important factors. The SLE Disease Activity Index (SLEDAI) was positively correlated with cMo and plasma cells (PC). The titer of anti-DNA antibodies was positively correlated with cMo, CXCR5- nB, and CXCR5- DNB. The C4 level was negatively correlated with CXCR5- DNB. The bioactivity of type I interferon was also positively correlated with these ICPs. Fever and renal involvement were associated with cMo. Rash was associated with CD8Tem and CXCR5- DNB. On the basis of the proliferative activity among five ICPs, SLE patients can be classified into five clusters showing different SLE phenotypes. Evaluation of the proliferative activity in each ICP can be linked to the clinical phenotypes of individual SLE patients and help in the treatment strategy.

SLE stratification based on BAFF and IFN-I bioactivity for biologics and implications of BAFF produced by glomeruli in lupus nephritis.

OBJECTIVE: B-cell activating factor (BAFF) is implicated in SLE pathogenesis. Blocking BAFF signalling has contributed to reducing glucocorticoid dosage and preventing organ damage. However, clinical characteristics of patients who may benefit from this therapy are not yet fully elucidated. Therefore, we identified patients with high BAFF-bioactivity to investigate their clinical characteristics and BAFF-producing cells. METHODS: We established the reporter cell for BAFF and investigated the clinical characteristics of SLE patients with high BAFF-bioactivity. We identified BAFF-expressing kidney cells using publicly available scRNA-seq data and immunohistological analysis. SLE patients were stratified based on the bioactivity of BAFF and type-I IFN (IFN-I) to identify associated characteristic clinical manifestations. RESULTS: SLE patients, especially patients with LN, had significantly higher serum BAFF-bioactivity than healthy controls (HC) and non-LN patients. Additionally, single-cell-RNA-seq data and immunohistological analysis of kidney samples from LN patients revealed that BAFF is expressed in glomerular macrophages and mesangial cells. Notably, BAFF bioactivity was elevated in the urine of LN patients compared with that of non-LN patients, while no IFN-I bioactivity was detected in the urine. Furthermore, SLE stratification based on bioactivities of serum BAFF and IFN-I revealed the clinical characteristics of patients: high BAFF represented patients with LN and high IFN-I represented patients with blood and skin manifestations. CONCLUSIONS: Monitoring urinary BAFF-bioactivity may be valuable in diagnosing LN. Furthermore, stratification based on serum BAFF and IFN-I bioactivities may allow the identification of appropriate patients for biologics targeting BAFF and IFN-I.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Safety and immunogenicity of a quadrivalent seasonal influenza vaccine adjuvanted with hydroxypropyl-ß-cyclodextrin: A phase 1 clinical trial.

OBJECTIVES: Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), an oligosaccharide used as an excipient in pharmaceutical preparation, was recently reported to function as a vaccine adjuvant to co-administered antigens. In this study, we investigated the safety and immunogenicity of a seasonal influenza vaccine adjuvanted with HP-ß-CyD (FluCyD-vac) in healthy adults compared with those of a standard seasonal influenza vaccine (Flu-vac). METHODS: We conducted a single-blinded randomized phase 1 clinical trial study, and used two quadrivalent split seasonal influenza vaccines: FluCyD-vac containing 9 µg of HA/strain and 20% w/v of HP-ß-CyD, and Flu-vac containing 15 µg of hemagglutinin (HA)/strain only. All participants were randomly assigned to receive a single dose of Flu/CyD-vac or Flu-vac at a ratio of 2:1. We assessed solicited and unsolicited adverse events (AEs) and immune responses using hemagglutination inhibition (HI) titers. In addition, we assessed T-cell function in peripheral blood mononuclear cells (PBMCs), after stimulation with HA vaccine strains, using flow cytometry. RESULTS: Among 36 healthy volunteers enrolled in the study (FluCyD-vac, n = 24; Flu-vac, n = 12), FluCyD-vac was well tolerated. Most of the solicited AEs were mild local skin reactions at the injection site. No serious AEs were reported in either group. HI titers 21 days after vaccination with FluCyD-vac were comparable with those of Flu-vac and sufficient to meet international criteria, despite reduced HA antigen doses. When PBMCs were stimulated with the four HA antigens in the vaccine, tumor necrosis factor (TNF)-α-producing CD4+ T cells were enhanced in the FluCyD-vac group. CONCLUSION: FluCyD-vac was well-tolerated and immunogenic, despite containing 40% less HA antigens than Flu-vac. This study showed that HP-ß-CyD is a potentially safe, novel adjuvant for human influenza vaccine. CLINICAL TRIAL REGISTRY: UMIN000028530.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

WT1 epitope-specific IgG and IgM antibodies for immune-monitoring in patients with advanced sarcoma treated with a WT1 peptide cancer vaccine.

The Wilms' tumor gene WT1 is highly expressed in various malignancies and may be a common target antigen for cancer immunotherapy. In our group, peptide-based cancer vaccines targeting WT1 CTL epitopes were developed as an immunotherapy for these malignancies. In the present study, WT1 epitope-specific immune responses were analyzed in 31 patients with advanced sarcoma with human leukocyte antigen-A*24:02- and WT1-expressing tumors who received the WT1-235 peptide vaccine as monotherapy. The serum levels of IgG and IgM antibodies against the target epitope WT1-235 and the non-target epitopes WT1-332 and WT1-271 were measured using ELISA. IgM antibodies against WT1-235, WT1-332 and WT1-271 were detected in three (9.6%), four (12.9%) and 20 patients (64.5%), respectively, prior to vaccine administration, indicating immune recognition of the WT1 antigen prior to administering the vaccine. Of 15 patients who had completed the 3-month treatment protocol, WT1-235 IgG was positive in five (33.3%) patients. An enzyme-linked immunospot assay revealed that WT1-235 epitope-specific IL-10 production/secretion in peripheral blood mononuclear cells declined in the first month of vaccine administration in all three patients with positivity for WT1-235 IgM at the start of the vaccine. Furthermore, positivity for both WT1-235 and WT1-271 IgM antibodies at the start of treatment was associated with unfavorable tumor control at 3 months after vaccine administration. These results suggested that WT1 epitope-specific IgG and IgM antibodies may be utilized as immune-monitoring markers for WT1 peptide cancer vaccine immunotherapy. The trials were entered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (https://www.umin.ac.jp/ctr; no. UMIN000002001 on May 24, 2009 and no. UMIN000015997 on December 20, 2014).

Cellular and Humoral Immune Responses Induced by an HLA Class I-restricted Peptide Cancer Vaccine Targeting WT1 Are Associated With Favorable Clinical Outcomes in Advanced Ovarian Cancer.

The HLA-A*24:02-restricted peptide vaccine targeting Wilms' tumor 1 (WT1) (WT1 vaccine) is a promising therapeutic strategy for ovarian cancer; however, its efficacy varies among patients. In this study, we analyzed WT1-specific immune responses in patients with advanced or recurrent ovarian cancer that was refractory to standard chemotherapies and their associations with clinical outcomes. In 25 patients, the WT1 vaccine was administered subcutaneously weekly for 3 months and biweekly thereafter until disease progression or severe adverse events. We assessed Wilms' tumor 1-specific cytotoxic T lymphocytes (WT1-CTLs) and Wilms' tumor 1 peptide-specific immunoglobulin G (WT1235-IgG). After vaccination, the percentage of tetramer high-avidity population of WT1-CTLs among CD8+ T lymphocytes (%tet-hi WT1-CTL) and the WT1235-IgG titer increased significantly, although the values were extremely low or below the limit of detection before vaccination (%tet-hi WT1-CTL: 0.003%-0.103%.; WT1235-IgG: <0.05-0.077 U/mL). Patients who had %tet-hi WT1-CTL of ≥0.25% (n=6) or WT1235-IgG of ≥0.10 U/mL (n=12) had a significantly longer progression-free survival than those of patients in the other groups. In addition, an increase in WT1235-IgG corresponded to a significantly longer progression-free survival (P=0.0496). In patients with systemic inflammation, as evidenced by elevated C-reactive protein levels, the induction of tet-hi WT1-CTL or WT1235-IgG was insufficient. Decreased serum albumin levels, multiple tumor lesions, poor performance status, and excess ascites negatively influenced the clinical effectiveness of the WT1 vaccine. In conclusion, the WT1 vaccine induced antigen-specific cellular and humoral immunity in patients with refractory ovarian cancer. Both %tet-hi WT1-CTL and WT1235-IgG levels are prognostic markers for the WT1 vaccine.

Identification of mouse helper epitopes for WT1-specific CD4+ T cells.

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L- effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Recovery from prolonged thrombocytopenia in patients with TAFRO syndrome: case series and literature review.

TAFRO syndrome is a newly proposed disease that is characterised by thrombocytopenia, anasarca, fever, reticulin fibrosis (or renal dysfunction), and organomegaly. Generally, high doses of corticosteroids are recommended for the initial treatment of TAFRO syndrome; however, some patients experience prolonged refractory thrombocytopenia after initiating such therapies. If corticosteroid treatment alone is ineffective, additional immunosuppressive therapies such as cyclosporine A are recommended. Since long-term use of immunosuppressive therapies with TAFRO syndrome sometimes causes serious infection, it is important to recognise the time to recovery from thrombocytopenia. In this study, we investigated how long it took to recover from thrombocytopenia, to aid clinicians in decision-making regarding the need to strengthen treatment for prolonged thrombocytopenia. Here, we describe three of our patients with TAFRO syndrome exhibiting prolonged thrombocytopenia. We also investigated the median period to recovery from this complication (defined as the time to increase the platelet count above 50,000/µL) after the initiation of high-dose corticosteroid treatment in our 3 cases and 38 peer-reviewed cases. We found that it took our patients 61 days to recover from thrombocytopenia; in comparison, our investigation of the 38 peer-reviewed case reports revealed a median recovery time of 47.5 days among previously reported patients. We showed the time to recovery from thrombocytopenia in patients with TAFRO syndrome for the first time. Our findings ought to be useful for decision-making among clinicians regarding the administration of other immunosuppressive treatments in addition to corticosteroid.

First-in-human randomised trial and follow-up study of Plasmodium falciparum blood-stage malaria vaccine BK-SE36 with CpG-ODN(K3).

BACKGROUND: BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan. METHODS: An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response. RESULTS: BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t-test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36. CONCLUSIONS: BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa. TRIAL REGISTRATION: JMACCT Clinical Trial Registry JMA-IIA00109.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Reader-free ELISPOT assay for immuno-monitoring in peptide-based cancer vaccine immunotherapy.

Cancer vaccine immunotherapy is a therapy that induces cellular immune responses against a target molecule to elicit clinical anti-tumor effects. These cellular immune responses against the target molecule are monitored to evaluate whether the antigen-specific cellular immune responses are induced and maintained during the vaccination period. Enzyme-linked immunospot (ELISPOT) assay is widely performed to analyze not only the frequency of immune cells, but also their effector functions as determined by their cytokine production/secretion. The present study aimed to develop a reader-free ELISPOT assay using a handy membrane-punching device termed ELI 8. With the assistance of particle analysis by ImageJ software, the results of spot counting were reproducible with high inter-assay and inter-examiner concordance. Immune cells that produce and secrete Th1 cytokines without antigen-peptide stimulation of peripheral blood mononuclear cells (PBMCs) were detected, and their frequencies in patients with cancer were significantly higher compared with those in healthy individuals. These frequencies varied between individuals, as well as between time points during the course of cancer vaccine immunotherapy in each patient. Due to the variability in spontaneous cytokine production/secretion by PBMCs, an antigen-specific immune response (IR) index is proposed, which is a ratio of the number of spot-forming cells (SFCs) subjected to antigen-stimulation to that of SFCs with spontaneous cytokine secretion without antigen-stimulation. This index may be used as a marker for antigen-specific cellular immune responses in patients treated with cancer immunotherapy. The IR index successfully detected the induction of Wilms' tumor 1-specific cellular immune responses in patients with cancer treated with cancer vaccine immunotherapy.

Immune adjuvant therapy using Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in advanced malignancies: A phase 1 study of safety and immunogenicity assessments.

The cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) is a bioactive component that is a strong immune adjuvant for cancer immunotherapy. BCG-CWS activates the innate immune system through various pattern recognition receptors and is expected to elicit antigen-specific cellular immune responses when co-administered with tumor antigens. To determine the recommended dose (RD) of BCG-CWS based on its safety profile, we conducted a phase I dose-escalation study of BCG-CWS in combination with WT1 peptide for patients with advanced cancer.The primary endpoint was the proportion of treatment-related adverse events (AEs) at each BCG-CWS dose. The secondary endpoints were immune responses and clinical effects. A BCG-CWS dose of 50, 100, or 200 µg/body was administered intradermally on days 0, 7, 21, and 42, followed by 2 mg of WT1 peptide on the next day. For the escalation of a dose level, 3 + 3 design was used.Study subjects were 18 patients with advanced WT1-expressing cancers refractory to standard anti-cancer therapies (7 melanoma, 5 colorectal, 4 hepatobiliary, 1 ovarian, and 1 lung). Dose-limiting toxicity occurred in the form of local skin reactions in 2 patients at a dose of 200 µg although no serious treatment-related systemic AEs were observed. Neutrophils and monocytes transiently increased in response to BCG-CWS. Some patients demonstrated the induction of the CD4 T cell subset and its differentiation from the naïve to memory phenotype, resulting in a tumor response.The RD of BCG-CWS was determined to be 100 µg/body. This dose was well tolerated and showed promising clinical effects with the induction of an appropriate immune response.