Circulating tumor-associated antigen-specific IFNγ+4-1BB+ CD8+ T cells as peripheral biomarkers of treatment outcomes in patients with pancreatic cancer.

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy.

Humoral and cellular immune responses to COVID-19 mRNA vaccines in immunosuppressed liver transplant recipients.

BACKGROUND: Liver transplant recipients (LTRs) are at a high risk of severe COVID-19 owing to immunosuppression and comorbidities. LTRs are less responsive to mRNA vaccines than healthy donors (HDs) or other immunosuppressed patients. However, the disruption mechanism in humoral and cellular immune memory responses is unclear. METHODS: We longitudinally collected peripheral blood mononuclear cells and plasma samples from HDs (n = 44) and LTRs (n = 54) who received BNT162b2 or mRNA-1273 vaccines. We measured the levels of anti-receptor-binding domain (RBD) antibodies and spike-specific CD4+ and CD8+ T-cell responses. RESULTS: Here, we show that the induction of anti-RBD IgG was weaker in LTRs than in HDs. The use of multiple immunosuppressive drugs is associated with lower antibody titers than only calcineurin inhibitor, and limits the induction of CD4+ T-cell responses. However, spike-specific CD4+ T-cell and antibody responses improved with a third vaccination. Furthermore, mRNA vaccine-induced spike-specific CD8+ T cells are quantitatively, but not qualitatively, limited to LTRs. Both CD4+ and CD8+ T cells react to omicron sublineages, regardless of the presence in HDs or LTRs. However, there is no boosting effect of spike-specific memory CD8+ T-cell responses after a third vaccination in HDs or LTRs. CONCLUSIONS: The third mRNA vaccination improves both humoral responses and spike-specific CD4+ T-cell responses in LTRs but provides no booster effect for spike-specific memory CD8+ T-cell responses. A third mRNA vaccination could be helpful in LTRs to prevent severe COVID-19, although further investigation is required to elicit CD8+ T-cell responses in LTRs and HDs.

People with a liver transplant don't have as strong an immune response to COVID-19 vaccines as healthy people. This study investigates how these individuals produce protective proteins, called antibodies, and CD4 and CD8 T cell immune responses. CD4 T cells are responsible for commanding the immune response and CD8 T cells for remembering and fighting the virus in future. We found that liver transplant recipients have a weaker ability to produce antibodies after vaccination, which is even more noticeable in those taking drugs to prevent transplant rejection. While a third vaccine dose improves their ability to produce antibodies, and to have a CD4 T cell response, it doesn't boost the CD8 T cell response. In summary, an extra vaccine dose can strengthen the immune response in liver transplant recipients but doesn't improve some aspects of their immune memory.

Hepatitis B surface antigen reduction is associated with hepatitis B core-specific CD8+ T cell quality.

Despite treatment, hepatitis B surface antigen (HBsAg) persists in patients with chronic hepatitis B (CHB), suggesting the likely presence of the virus in the body. CD8+ T cell responses are essential for managing viral replication, but their effect on HBsAg levels remains unclear. We studied the traits of activated CD8+ T cells and HBV-specific CD8+ T cells in the blood of CHB patients undergoing nucleos(t)ide analog (NUC) therapy. For the transcriptome profiling of activated CD8+ T cells in peripheral blood mononuclear cells (PBMCs), CD69+ CD8+ T cells were sorted from six donors, and single-cell RNA sequencing (scRNA-seq) analysis was performed. To detect HBV-specific CD8+ T cells, we stimulated PBMCs from 26 donors with overlapping peptides covering the HBs, HBcore, and HBpol regions of genotype A/B/C viruses, cultured for 10 days, and analyzed via multicolor flow cytometry. scRNA-seq data revealed that CD8+ T cell clusters harboring the transcripts involved in the cytolytic functions were frequently observed in donors with high HBsAg levels. Polyfunctional analysis of HBV-specific CD8+ T cells utilized by IFN-γ/TNFα/CD107A/CD137 revealed that HBcore-specific cells exhibited greater polyfunctionality, suggesting that the quality of HBV-specific CD8+ T cells varies among antigens. Moreover, a subset of HBcore-specific CD8+ T cells with lower cytolytic potential was inversely correlated with HBsAg level. Our results revealed a stimulant-dependent qualitative difference in HBV-specific CD8+ T cells in patients with CHB undergoing NUC therapy. Hence, the induction of HBcore-specific CD8+ T cells with lower cytolytic potential could be a new target for reducing HBsAg levels.

The impact of cellular senescence and senescence­associated secretory phenotype in cancer­associated fibroblasts on the malignancy of pancreatic cancer.

Cancer­associated fibroblasts (CAFs) are implicated in the strong malignancy of pancreatic cancer (PC). Various CAF subtypes have different functions, and their heterogeneity likely influence the malignancy of PC. Meanwhile, it is known that senescent cells can create a tumor­promoting microenvironment by inducing a senescence­associated secretory phenotype (SASP). In the present study, the effects of individual differences in CAFs on PC malignancy were investigated with a focus on cellular senescence. First, primary cultures of CAFs from 8 PC patients were generated and co­cultured with PC cell lines. This co­culture assay showed that differences in CAFs induce differences in PC cell proliferation. It was further investigated which clinical factors affected the malignant potential of CAF and it was found that the difference of malignant potential of each CAF was marginally related to the age of original patients. Next, to verify the senescence of CAFs really affected the malignant potential of CAF, PCR array analysis of each CAF sample was performed and it was revealed that expression of genes about cellular senescence and SASP such as tumor protein p53, nuclear factor kappa B subunit 1, and IL6, are related to the malignant potential of CAFs impacting on PC proliferation. Finally, to elucidate the effect of p53­mediated cellular senescence of CAFs on malignant potential of PC, it was examined whether CAFs with the treatment of p53 inhibitor affected PC cell proliferation in co­culture assays. The treatment of CAFs with p53 inhibitor significantly suppressed PC cell proliferation. In addition, a comparison of the concentration of IL­6, a SASP cytokine, in the co­culture supernatant showed a significant decrease in the sample after p53 inhibitor treatment. In conclusion, the present results suggested that proliferation potential of PC may be related to p53­mediated cellular senescence and SASP of CAFs.

Elucidation of the role of nucleolin as a cell surface receptor for nucleic acid-based adjuvants.

Nucleic acid-based adjuvants such as CpG oligonucleotides (CpG ODNs) and poly(I:C) are potential vaccine adjuvants for infectious diseases and cancers. However, the mechanism by which their cell surface receptors promote their uptake into dendritic cells (DCs) and shuttle them to intracellular Toll-like receptors remains to be further investigated. Here, we demonstrated a role for nucleolin, a multifunctional DNA- and RNA-binding protein and a major constituent of the nucleolus, as one of the cell-surface receptors for nucleic acid-based adjuvants. Nucleolin on mouse DC surface bound directly to A-type CpG ODN, B-type CpG ODN, and poly(I:C) and promoted their internalization into cells following DC maturation in vitro. In human DCs, nucleolin also contributed to the binding and internalization of both types of CpG ODNs and subsequent cytokine production. Furthermore, nucleolin played a crucial role in cytokine production and activating antigen-specific antibodies and T cell responses induced by B-type CpG ODN in vivo in mice. Our findings provide valuable information that can help improve the efficacy and safety of these adjuvants.

OMIP 078: A 31-parameter panel for comprehensive immunophenotyping of multiple immune cells in human peripheral blood mononuclear cells.

This 31-parameter panel was developed for simultaneously measuring multiple immune cell populations including T cells, B cells, natural killer cells, dendritic cells, monocytes, and hematopoietic progenitor cells in human peripheral blood mononuclear cells. This panel enables the capture of individual immune dynamics and assessments of single-cell changes in the immune system that are associated with aging and diseases. This panel includes markers to separate the differentiation status of each cell population and might be applicable to studies of infectious and autoimmune diseases, as patient samples are usually limited in volume and require an analysis system that provides a relatively large amount of information.

Simultaneous monitoring assay for T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface.

Isolation of antigen (Ag)-specific T cells is an important step in the investigation of T-cell immunity. Activation-induced markers (AIMs), such as CD154/tumor necrosis factor (TNF)/CD107A/CD134/CD137 enable the sorting of Ag-specific T cells without using human leukocyte antigen (HLA)-multimers. However, optimal conditions suitable for simultaneous detection of both Ag-specific CD4 and CD8 T cells have not been investigated. Here, conditions were optimized to simultaneously detect the maximum number of activated CD4 and CD8 T cells in a TCR-dependent manner. First, the frequency of total pools of AIM-positive cells induced by superantigen, staphylococcal enterotoxin B (SEB), stimulation in various culture conditions was monitored and compared side-by-side. The total amount of AIM-positive CD4 T cells, but not CD8 T cells, was significantly abrogated by addition of brefeldin A. TNF-alpha converting enzyme inhibitor treatment effectively increased the TNF-positive population, without affecting other markers' positivity. AIM-positive CD4 T cells and CD8 T cells were detected at least 3 h after stimulation. Furthermore, examination of the multiple combination of each marker revealed that minimum contribution of CD134 on the total pool of AIM-positive cells at this setting, suggesting the essential and non-essential AIMs to maximize the detected number of AIM-positive cells. Taken together, this optimized method will be a useful tool for the simultaneous monitoring the T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface including Ag-specific T cells.

[A Case of Duodenum Neuroendocrine Tumor with Multiple Liver Metastasis Treated with Multimodal Therapy].

The patient is a 65-year-old woman with anemia. The multiple liver tumors detected by ultrasonography, it was diagnosed as neuroendocrine tumor(NET), G2 by biopsy. There was an ulcer at the bulb of the duodenum, so we diagnosed liver metastasis of duodenum NET. Because the liver tumors spreaded to both right and left lobes, we carrying out a transcatheter arterial embolization(TAE)twice to liver metastasis, and chemotherapy by octreotide was performed. 20 months after the beginning of treatment, a 4 cm tumor was remained in the left lobe but others were not detected by computed tomography, so we performed cytoreductive surgery. Duodenum bulb resection and left hepatectomy was performed and the specimens were NET, G2 in the pathological findings. We detected a lot of tumors less than 1 cm in the right lobe during the operation, so TAE was carried out for the right lobe after surgery. The disease showed no progression for 28 months after the first admission(post operation5 months).

Anti-metastatic Effect of Short-term Postoperative Anticoagulation for Patients Undergoing Curative Resection of Colorectal Cancer.

BACKGROUND: The purpose of this study was to examine the efficacy of anticoagulant fondaparinux postoperatively for preventing recurrence after curative resection of colorectal cancer with lymphnode metastasis. PATIENTS AND METHODS: The records of 279 patients who underwent curative resection of colorectal cancer with lymph-node metastasis between 2006 and 2013 were reviewed. Patients were divided into two groups based on the type of prophylaxis for postoperative venous thromboembolism: the FPX group, treated with subcutaneous fondaparinux plus intermittent pneumatic compression; and the IPC group, treated with intermittent pneumatic compression alone. Recurrence-free survival was compared using propensity score matching. RESULTS: In the propensity score-matched cohort, the 3-year recurrence-free survival rate was 74.9% and 74.4% in the FPX (n=61) and IPC groups (n=61), respectively (p=0.830). CONCLUSION: Our results do not suggest that short-term postoperative anticoagulation as prophylaxis for venous thromboembolism prevents colorectal cancer recurrence after curative resection.

[A Case of Recurrent Breast Cancer with Adrenal Metastasis Resected Using Laparoscopic Surgery].

We report a case of recurrent breast cancer with involvement of the right adrenal grand, which was resected using laparoscopic surgery. The patient was a 41-year-old woman who underwent duct-lobular segmentectomy for ductal carcinoma in situ. Histopathology showed microinvasion, and tested positive for ER and PR, but negative for HER2, so we applied radiation to the remaining breast and administered tamoxifen. After 9 months, local recurrence was detected and quadrantectomy with axillary lymph node dissection was performed. One year and 10 months later, local recurrence was again detected and a tumorectomy was performed. The adjuvant therapy was changed to an LH-RH analog plus anastrozole and it was administered effectively for 5 years. Left ileal metastasis appeared in the 2nd month after completion of the adjuvant chemotherapy, so radiation was applied and an LH-RH analog plus exemestane administration was started. Three years passed without recurrence, but a right adrenal tumor appeared on computed tomography. The tumor grew over 6 months, so laparoscopic right adrenalectomy was performed. Histopathologically, the tumor tested positive for ER and PR, and negative for HER2 so we diagnosed metastasis of breast cancer, and administered an LH-RH analog plus exemestane. The patient's disease has not progressed in the 3 months since surgery.

[A Case of Combined Modality Therapy for a Cervical Lymph Node Recurrence after Surgery for Esophageal Cancer].

In the present report, we describe a man with type 2 progressive squamous cell carcinoma (cT3N1M0, cStage Ⅲ) that was detected in the esophago-gastric junction during follow-up after ESD for early gastric cancer. We performed a middle inferior part esophagectomy, a 2-region dissection, and a posterior mediastinum gastric tube reconstruction after preoperative chemotherapy (docetaxel plus cisplatin plus 5-FU). The patient only received 1 course of preoperative chemotherapy because of neutropenia. The pathology results were pT3N2M0, pStage Ⅲ. Six months later, we started chemotherapy (nedaplatin plus adriamycin plus 5-FU) owing to an abdominal lymph node recurrence. We administered 3 courses, but then switched to radiotherapy because of AEs. After receiving a radiation dose of 50.4 Gy, the patient experienced a para-aortic lymph node recurrence and was administered 50.4 Gy for the new lesion, resulting in a CR. Six months later, we identified lymph node recurrences under the left superficialis neck muscle and performed left cervical lymph node resection. All 3 of the enlarged lymph nodes that we resected were found to contain a metastasis of esophageal cancer. Currently (after 6 months), there are no signs of recurrence.