Engineered anti-PDL1 with IFNα targets both immunoinhibitory and activating signals in the liver to break HBV immune tolerance.

OBJECTIVE: The purpose of this study is to develop an anti-PDL1-based interferon (IFN) fusion protein to overcome the chronic hepatitis B virus (HBV)-induced immune tolerance, and combine this immunotherapy with a HBV vaccine to achieve the functional cure of chronic hepatitis B (CHB) infection. DESIGN: We designed an anti-PDL1-IFNα heterodimeric fusion protein, in which one arm was derived from anti-PDL1 antibody and the other arm was IFNα, to allow targeted delivery of IFNα into the liver by anti-PDL1 antibody. The effect of the anti-PDL1-IFNα heterodimer on overcoming hepatitis B surface antigen (HBsAg) vaccine resistance was evaluated in chronic HBV carrier mice. RESULTS: The anti-PDL1-IFNα heterodimer preferentially targeted the liver and resulted in viral suppression, the PD1/PDL1 immune checkpoint blockade and dendritic cell activation/antigen presentation to activate HBsAg-specific T cells, thus breaking immune tolerance in chronic HBV carrier mice. When an HBsAg vaccine was administered soon after anti-PDL1-IFNα heterodimer treatment, we observed strong anti-HBsAg antibody and HBsAg-specific T cell responses for efficient HBsAg clearance in chronic HBV carrier mice that received the combination treatment but not in those that received either single treatment. CONCLUSIONS: Targeting the liver with an engineered anti-PDL1-IFNα heterodimer can break HBV-induced immune tolerance to an HBsAg vaccine, offering a promising translatable therapeutic strategy for the functional cure of CHB.

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single-cell RNA sequencing (scRNA-seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue-resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function-related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA-seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal-foetal interface by regulating the activity of NK cells.

Cytochrome P450 26A1 modulates natural killer cells in mouse early pregnancy.

Cytochrome P450 26A1 (CYP26A1) has a spatiotemporal expression pattern in the uterus, with a significant increase in mRNA and protein levels during peri-implantation. Inhibiting the function or expression of CYP26A1 can cause pregnancy failure, suggesting an important regulatory role of CYP26A1 in the maintenance of pregnancy. However, little is known about the exact mechanism involved. In this study, using a pCR3.1-cyp26a1 plasmid immunization mouse model and a Cyp26a1-MO (Cyp26a1-specific antisense oligos) knockdown mouse model, we report that the number of Dolichos biflorus agglutinin (DBA) lectin-positive uterine natural killer (uNK) cells was reduced in pCR3.1-cyp26a1 plasmid immunized and Cyp26a1-MO-treated mice. In contrast, the percentage of CD3- CD49b+ NK cells in the uteri from the treatment group was significantly higher than that of the control group in both models. Similarly, significantly up-regulated expression of CD49b (a pan-NK cell marker), interferon gamma, CCL2, CCR2 (CCL2 receptor) and CCL3 were detected in the uteri of pCR3.1-cyp26a1- and Cyp26a1-MO-treated mice. Transcriptome analysis suggested that CYP26A1 might regulate NK cells through chemokines. In conclusion, the present data suggest that silencing CYP26A1 expression/function can decrease the number of uNK cells and significantly increase the percentage of CD3- CD49b+ NK cells in the uteri of pregnant mice. These findings provide a new line of evidence correlating the deleterious effects of blocking CYP26A1 in pregnancy with the aberrant regulation of NK cells in the uterus.

The Balance between Conventional DCs and Plasmacytoid DCs Is Pivotal for Immunological Tolerance during Pregnancy in the Mouse.

Dendritic cells (DCs), which can shape their functions depending on the microenvironment, are crucial for the delicate balance of immunity and tolerance during pregnancy. However, the mechanism underlying the microenvironment-educated plasticity of DC differentiation during pregnancy remains largely unclear. Here, we found that the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) is regulated in a tissue-specific manner during pregnancy. The ratio of cDCs and pDCs remained constant in the spleen. However, the ratio changed in the para-aortic lymph nodes (LNs), where cDC percentages were significantly reduced concurrent with an increase in pDCs from E8.5 to E16.5. Moreover, the expansion of pDCs and T regulatory (Treg) cells was correlated in the para-aortic LNs, and pDCs had more potential to induce regulatory T cells (Tregs) compared with cDCs (independent of IDO expression). Notably, the balance between cDCs and pDCs is disrupted in IFN-γ-induced abnormal pregnancy, accompanied by lower Treg percentages in the para-aortic LNs and decidua. To further identify the underlying mechanism, we found that elevated IFN-γ can increase the levels of GM-CSF to alter the differentiation of pDCs into cDCs in vivo. Therefore, we provide a novel regulatory mechanism underlying pregnancy-related immune tolerance that involves the balance of DC subsets, which may offer a new target for the prevention of human spontaneous abortion.

Seminal plasma induces inflammation in the uterus through the γδ T/IL-17 pathway.

After insemination, a large number of leukocytes migrate into the uterus, which is accompanied by intense inflammation. However, the details of how seminal plasma interacts with the uterus are still not very clear. Here, we present that neutrophils migrate and accumulate around the uterine epithelium following insemination, which is accompanied by an increase in interleukin (IL) 17A levels. Additionally, we find that γδ T cells are the major source of IL-17A, and the seminal plasma could induce the γδ T cells to secret IL-17A. Blocking IL-17A could reduce the number of neutrophils in the uterus and prevent them from migrating to the epithelium by decreasing the chemokines CXCL1, CXCL2 and CXCL5. Blocking IL-17A did not affect the Th1/Th2 balance but actually diminished the inflammation in the uterus by reducing the expression of IL-1ß and TNF-α. In summary, we found a new mechanism by which seminal plasma could influence the inflammation in the uterus through the γδ T/IL-17 pathway to regulate the expression of various chemokines and cytokines.

IFN-γ modulates Ly-49 receptors on NK cells in IFN-γ-induced pregnancy failure.

We have previously shown that interferon gamma (IFN-γ) induces aberrant CD49b(+) natural killer (NK) cell recruitment by regulating CX3CL1 and eventually provokes foetal loss. In this study, we show that IFN-γ also modulates Ly-49 receptors on NK cells during pregnancy failure. The percentages of Ly-49A(+) and Ly-49G2(+) NK cells in the uteri of the IFN-γ-treated group were significantly lower than those observed in the control group. Moreover, the median fluorescence intensity (MFI) values of Ly-49A and Ly-49G2 expression on NK cells in the uteri of the IFN-γ-treated group were significantly lower than those of the control group. Using isolated spleen leucocytes, we further found that IFN-γ significantly reduced the percentage of Ly-49A(+) NK cells in vitro. However, CX3CL1 was not involved in the modulation of Ly-49 receptors, and the expression of CX3CR1 was not regulated by IFN-γ in spleen leucocytes. Collectively, our data indicate that IFN-γ can modulate Ly-49 receptors on NK cells and this process may play a role in IFN-γ-induced pregnancy failure. Thus, we provide a new line of evidence correlating the deleterious effects of IFN-γ with its role in regulating NK cell Ly-49 receptors during pregnancy failure.