Neoantigen vaccination augments antitumor effects of anti-PD-1 on mouse hepatocellular carcinoma.

Immune checkpoint inhibitors are groundbreaking resources for cancer therapy. However, only a few patients with hepatocellular carcinoma (HCC) have shown positive responses to anti-PD-1 therapy. Neoantigens are sequence-altered proteins resulting from somatic mutations in cancer. This study identified the neoantigens of Hep-55.1C and Dt81 Hepa1-6 HCCs by comparing their whole exome sequences with those of a normal C57BL/6 mouse liver. Immunogenic long peptides were pooled as peptide vaccines. The vaccination elicited tumor-reactive immune responses in C57BL/6 mice, as demonstrated by IFN-γ ELISPOT and an in vitro killing assay of splenocytes. In the treatment of three mouse HCC models, combined neoantigen vaccination and anti-PD-1 resulted in more significant tumor regression than monotherapies. Flow cytometry of the tumor-infiltrating lymphocytes showed decreased Treg cells and monocytic myeloid-derived suppressor cells, increased CD8+ T cells, enhanced granzyme B expression, and reduced exhaustion-related markers PD-1 and Lag-3 on CD8+ T cells in the combination group. These findings provide a strong rationale for conducting clinical studies of using neoantigen vaccination in combination with anti-PD-1 to treat patients with HCC.

In situ vaccination followed by intramuscular poly-ICLC injections for the treatment of hepatocellular carcinoma in mouse models.

The efficacy of treatment for advanced hepatocellular carcinoma (HCC) has remained limited. Polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) is a synthetic double-stranded RNA that serves as a viral mimic and induces an immune response. Intratumoral (IT) poly-ICLC injections can induce an autovaccination effect and prime the immune system, whereas intramuscular (IM) injection of poly-ICLC can attract and maintain tumor-specific cytotoxic T lymphocytes in tumors. We found that IT injection of poly-ICLC upregulated the expression of CD83 and CD86 on conventional type 1 dendritic cells in tumors. Combination therapy with IT followed by IM injections of poly-ICLC significantly inhibited tumor growth and increased the tumor-infiltrating CD8+ T cells in two syngeneic mouse models of HCC. Depletion of CD8+ T cells attenuated the antitumor effect. An IFN-γ enzyme-linked immunospot of purified tumoral CD8+ T cells revealed a significant proportion of tumor-specific T cells. Finally, the sequential poly-ICLC therapy induced abscopal effects in two dual-tumor models. This study provides evidence that the sequential poly-ICLC therapy significantly increased infiltration of tumor-specific CD8+ T cells in the tumors and induced CD8+ T cell-dependent inhibition of tumor growth, as well as abscopal effects.

Therapeutic Effects of Anti-PD1 Immunotherapy on Hepatocellular Carcinoma Under Administration of Tacrolimus.

BACKGROUND: Liver transplantation (LT) is the treatment of choice for patients with hepatocellular carcinoma (HCC). Recurrence of HCC after LT occurs in 10% to 20% of cases. Preclinical studies to evaluate immune checkpoint inhibitors in conjunction with immunosuppressant treatment in transplant recipients have been lacking. Here, we evaluated the efficacy, safety, and mechanism of programmed cell death-1 (PD1) blockade under tacrolimus treatment in transplant recipients. METHODS: We used a murine allogeneic skin transplantation model and murine syngeneic subcutaneous and orthotopic HCC models and measured the tumor volume and the change in tumor-infiltrating lymphocytes under PD1 blockade and tacrolimus treatment. RESULTS: Tacrolimus treatment prolonged allograft survival in the allogeneic transplantation model and enhanced tumor growth in both subcutaneous and orthotopic HCC models. PD1 blockade suppressed tumor growth and lung metastasis in correlation with the number of infiltrating CD8 + T cells. Under tacrolimus treatment, PD1 blockade still resulted in an antitumor effect accompanied by a significant increase in tumor-infiltrating CD8 + T cells, natural killer cells, dendritic cells, and natural killer T cells. Tacrolimus treatment rescued the acceleration of transplant rejection induced by PD1 blockade in the allogeneic transplantation model. CONCLUSIONS: Our data suggest that treatment with high-dose tacrolimus in conjunction with PD1 blockade has an antitumor effect and reduces transplant rejection in mouse models of allograft skin transplantation and HCC. Thus, these results suggest that a clinical trial of PD1 inhibitors for HCC in LT merits consideration.

The role of Klebsiella pneumoniae rmpA in capsular polysaccharide synthesis and virulence revisited.

Klebsiella pneumoniae community-acquired pyogenic liver abscess (PLA) is an emerging infectious disease. The rmpA gene (for regulator of mucoid phenotype A) has been reported to be associated with PLA in prevalence studies. NTUH-K2044, a K1 PLA isolate, carries three rmpA/A2 genes: two large-plasmid-carried genes (p-rmpA and p-rmpA2) and one chromosomal gene (c-rmpA). In this study, we re-examined the role of rmpA/A2 in PLA pathogenesis to clarify the relationship of rmpA/A2 and capsular serotype to virulence. Using isogenic gene deletion strains and complemented strains of NTUH-K2044, we demonstrated that only p-rmpA enhanced expression of capsular polysaccharide synthesis (cps) genes and capsule production. Nevertheless, the lethal dose and in vivo competitive index indicated that p-rmpA does not promote virulence in mice. The prevalence of these three rmpA/A2 and capsular types in 206 strains was investigated. This revealed a correlation of rmpA/A2 with six PLA-related capsular types (K1, K2, K5, K54, K57 and KN1). However, the correlation of rmpA/A2 with K1 strains from the West was less obvious than with the strains from Asia (17/22 vs 39/39, P = 0.0019). Among the three rmpA/A2 genes, p-rmpA was the most prevalent. Due to the strong correlation with PLA-related capsular types, p-rmpA could serve as a surrogate marker for PLA. We found an association of p-rmpA with three widely spaced loci in a large plasmid (30/32). Therefore, rmpA could be co-inherited together with virulence genes carried by this plasmid.

Aboriginal Taiwanese hepatitis B carriers have more favorable viral factors than Han Chinese carriers.

Several viral factors are associated with disease progression in hepatitis B virus (HBV) carriers. Compared with Taiwanese Han Chinese, Taiwanese aborigines have a higher prevalence of chronic HBV infection and a higher standardized mortality rate of chronic liver diseases but a lower standardized mortality rate of hepatocellular carcinoma (HCC). The aim of this study was to investigate whether aboriginal Taiwanese HBV carriers have more favorable viral factors which reduce the risk for HCC than Han Chinese carriers. Blood samples from 3,488 HBV carriers (1,527 aborigines and 1,961 Han Chinese) were assayed for aminotransferases, hepatitis B e antigen (HBeAg), HBV DNA, and HBV genotype. Aboriginal HBV carriers had a lower HBeAg-positive rate (5.3% vs. 10.2%, P < 0.0001) and a lower viral load of HBV DNA > 2,000 IU/ml (27.4% vs. 36.7%, P < 0.0001) but a higher rate of alcohol consumption (40.0% vs. 19.3%, P < 0.0001) than Han Chinese carriers. The prevalence of HBV genotype B in aboriginal carriers (92.7%) was significantly higher than that in Han Chinese carriers (72.7%) in all age groups (P < 0.05). In addition, patients with rare genotype D infections were clustered in a township in southern Taiwan. In conclusion, aboriginal Taiwanese HBV carriers have more favorable viral factors than Han Chinese carriers, which may be partly responsible for the lower standardized mortality rate of HCC in Taiwanese aborigines.

Alterations of tumour suppressor gene PPP2R1B in hepatocellular carcinoma.

To evaluate whether the tumour suppressor gene, PPP2R1B, is involved in pathogenesis of hepatocellular carcinoma (HCC), reverse transcription-polymerase chain reaction (RT-PCR) and cDNA sequencing were performed. Eleven of 38 (29%) tumours and 1 of 34 (3%) corresponding non-tumour tissues showed coexpression of wild-type and aberrant mRNA. Various deletions were found in aberrant transcripts. Southern blot analysis did not show gene deletion in tumours, suggesting abnormal RNA splicing may be involved. These data suggest the possibility that aberrant transcripts of PPP2R1B might be associated with the development of HCC.

Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection.

Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. To identify genes associated with liver infection, we used a DNA microarray to compare the transcriptional profiles of three strains causing liver abscess and three strains not associated with liver infection. There were 13 clones that showed higher RNA expression levels in the three liver infection strains, and 3 of these 13 clones contained a region that was absent in MGH 78578. Sequencing of the clones revealed the replacement of 149 bp of MGH 78578 with a 21,745-bp fragment in a liver infection strain, NTUH-K2044. This 21,745-bp fragment contained 19 open reading frames, 14 of which were proven to be associated with allantoin metabolism. The K2044 (DeltaallS) mutant showed a significant decrease of virulence in intragastric inoculation of BALB/c mice, and the prevalence of this chromosomal region was significantly higher in strains associated with liver abscess than in those that were not (19 or 32 versus 2 of 94; P = 0.0001 [chi(2) test]). Therefore, the 22-kb region may play a role in K. pneumoniae liver infection and serve as a marker for rapid identification.