Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor superfamily, inhibits T cell activity by providing a second signal to T cells in conjunction with signaling through the T-cell receptor. PD-1/PD-1 ligand (PD-L) signaling system is indicated to be involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We hypothesized that this signaling system is also involved in the pathogenesis of Hodgkin lymphoma (HL). We examined expression of B7-H1 and B7-DC, two known PD-Ls, in lymphoid cell lines using RT-PCR and flow cytometry. They were expressed in HL and several T-cell lines, whereas most B-NHL lines lacked their expression. Immunohistochemical staining of HL tissues demonstrated that PD-Ls were also expressed in primary H/RS cells. As gene expression of B7-H1 and B7-DC was increased in Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell lines, we examined the effect of EBV latent membrane proteins on their gene regulation. By luciferase reporter assay, both LMP1 and LMP2A were shown to enhance promoter activity of B7-H1 and B7-DC genes. This finding implies that in cases of EBV-positive HL, latent membrane proteins may help H/RS cells escape from host immune surveillance by upregulating PD-L gene expression. We next analyzed PD-1 expression of tumor-infiltrating T cells of HL tissue samples by flow cytometry, and found that PD-1+ cells were elevated markedly in these cells. As HL patients are well recognized as having defective cellular immunity, we compared PD-1 expression level in peripheral blood T cells of HL patients with those of healthy volunteers and B-NHL patients. PD-1 was significantly elevated in peripheral T cells of HL patients compared to the other two groups. PD-1+ T cells were highest in patients with active disease, and tended to decline along with treatment. Although regulatory T cells are reported to play a part in the pathogenesis of HL, FOXP3+ T cells were not significantly elevated in peripheral T cells of HL patients, and PD-1+ T cells did not overlap with these regulatory population. To elucidate whether the PD-1/PD-L signaling pathway is functional in the immunosuppressive microenvironment of HL, we finally examined the effect of blockade of this pathway. After culturing bulk HL tumor cells with anti-PD-L blocking antibodies, IFN-γ production was measured by ELISA. Blockade of PD-Ls augmented IFN-γ production of HL-infiltrating T cells. We concluded that anti-tumor activity of HL-infiltrating T cells was inhibited via the PD-1/PD-L pathway, and this inhibition could be successfully relieved by PD-L blockade. Taken together, our observations indicate that “T-cell exhaustion” is essential to the pathogenesis of HL, and tumor-infiltrating T cells around H/RS cells seem to be kept in balance by this inhibitory signaling. Our findings provide a potentially effective and clinically applicable strategy for the immunotherapy of HL.

Antibody-mediated rejection (AMR) is a refractory rejection after ABO blood-type incompatible (ABOi) or donor-specific antibody (DSA)-positive liver transplantation (LT). Pretransplant rituximab desensitization dramatically reduced posttransplant AMR development; however, risk factors for AMR in the rituximab era remain unclear in both ABOi living-donor LT (ABOi-LDLT) and preformed DSA-positive LT (pDSA-LT). Of our 596 adult LDLTs (≥18 y) after rituximab introduction (2004–2019), 136 were ABOi-LDLT (22.8%). After excluding retransplants (9), acute liver failure (7), and protocol deviations (16), 104 ABOi-LDLTs were finally enrolled. Of these, 19 recipients developed AMR, 18 of which occurred within 2 weeks after transplantation (95%). ABOi-AMR significantly worsened graft and recipient survival than those without (p=0.02 and 0.04, respectively). Model for End-stage Liver Disease (MELD) ≤13 (OR: 5.15 [1.63–16.3], p=0.005) and pre-rituximab anti-ABO IgM-titer ≥128 (OR: 3.25 [1.05–10.0], p=0.03) were identified as independent risk factors for ABOi-AMR development. Recipients fulfilling both factors showed significantly worse survival rates than those who did not (p=0.003). Of 352 adult LTs, after introducing the LABScreen Single Ag method (2009–2019), pDSA with mean fluorescence intensity (MFI) ≥500 was detected in 50 cases (14.2%). After excluding 10 ABOi-LDLTs, 40 pDSA-LTs were finally analyzed, of which 5 developed AMR. The combination of high-titer (sum-MFI ≥10,000) and multi-loci pDSAs was a significant risk factor for pDSA-AMR development (p <0.001); however, it did not affect the 5-year recipient survival compared with those without (p=0.56). In conclusion, preoperative MELD ≤13 and pre-rituximab anti-ABO IgM-titer ≥128 for ABOi-LDLT, and the combination of sum-MFI ≥10,000 and multi-loci pDSAs for pDSA-LT, are risk factors for AMR in the era of rituximab desensitization. Characteristically, ABOi-AMR significantly deteriorated graft and recipient survival, whereas pDSA-AMR did not.

[Background] Epigenetic and genetic characteristics in Japanese follicular lymphoma (FL) patients have been scarcely evaluated. EHZ2 represents a new therapeutic target in FL and identifying candidates for EZH2 inhibition medication in the general hospital settings without access to genetic sequencing is critical. Since EZH2 regulates methylation at the 27th amino acid in Histone (H3K27), we evaluated the characteristics of EZH2 expression and H3K27 methylation in the Japanese FL cohort as well as their association with genetic characteristics. Prognostic impact of these factors on clinical outcomes was also examined. [Method and Patients] A total of 164 patients (female, n=91; male, n=73) diagnosed with FL at Kyoto University Hospital between 2005 and 2020 were included. A total of 105 patients were assessed immunohistochemically using H-score for EZH2 expression (EZH2-score) and H3K27me3/me2 scoring (Methylation-score). Impact of these scores at diagnosis on overall survival (OS) and progression/relapse after the 1st immunochemotherapy were examined. We also performed next generation sequencing for 357 genes for 116 samples at diagnosis and examined their correlation with immunohistochemical characteristics. [Results] Median age at diagnosis was 61 years (range, 33-86) and the median follow-up years was 7.5. Median EZH2-score was 70 (range, 0-290) and median Methylation-score was 2.38 (range, -4.25-4.95), with no apparent correlation. Twenty-five patients (21.6%) harbored EZH2 mutations at diagnosis, all of which were in the mutation hotspots and showed a strong correlation with high Methylation-scores (p-value &lt; 0.001). Based on the prognostic impact of Methylation- and EZH2-score on survival, patients were divided into 3 groups: Group I, those with low Methylation-scores (&lt;2) and high EZH2-scores (≥100); Group II, those with low Methylation-scores (&lt;2) and low EZH2-scores (&lt;100); Group III, those with high Methylation scores (≥100). Group I showed inferior OS compared to Group III (p=0.038) with high risk of rapid relapse/progression (62.5%) within 2 years, who frequently harbored mutations in CSMD3 (vs Group III: p=0.004), KMT2D (p&lt;0.001) and SETD1B (p&lt;0.001). Distribution of mutated genes among Group III patients differed according to the existence of EZH2 mutation, with high frequency of mutated USH2A (p=0.019) and MGAM (p=0.019) genes in those with EZH2 mutation, whereas mutations in TP53 were observed only in those without (p=0.072). [Conclusion] Methylation-score could be helpful in distinguishing patients with and without EZH2 mutations and enables clinicians to identify candidates for EZH2 inhibition medications. Combination of EZH2- and Methylation-scores predicts clinical outcomes after the 1st immunochemotherapy, which also could potentially identify genetically distinct population in the heterogeneous FL cohort. Citation Format: Mizuki Watanabe, Naoki Goda, Junya Kanda, Akihiko Yoshizawa, Momoko Nishikori, Yasuhito Nannya, Kenichi Yoshida, Seishi Ogawa, Hironori Haga, Akifumi Takaori-Kondo. Epigenetic and genetic characteristics and their association with prognosis of follicular lymphoma: Analysis at a Japanese single institution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1002.