Yu Zhou

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Immune checkpoint molecules have been identified as crucial regulators of the immune response, which motivated the emergence of immune checkpoint-targeting therapeutic strategies. However, the prognostic significance of the immune checkpoint molecules PD-1, CTLA4, TIM-3 and LAG-3 remains controversial. The aim of our study was to conduct a systematic assessment of the expression of these immune checkpoint molecules across different cancers in relation to treatment response, tumor-infiltrating immune cells and survival. Oncomine and PrognoScan database analyses were used to investigate the expression levels and prognostic values of these immune checkpoint molecule genes across various cancers. Then, we used Kaplan-Meier plotter to validate the associations between the checkpoint molecules and cancer survival identified in the PrognoScan analysis. TIMER analysis was used to evaluate immune cell infiltration data from The Cancer Genome Atlas. Finally, we used Gene Expression Profiling Interactive Analysis to investigate the prognostic value of these four checkpoint molecules and assess the correlations between these four checkpoint molecules and genetic markers. These immune checkpoint molecules may potentially serve as prognostic factors and therapeutic targets in breast cancer, ovarian cancer and lung cancer. The prognostic roles of these checkpoint molecules varied greatly across cancers, which implied a noteworthy amount of heterogeneity among tumors, even within the same molecular subtype. In addition, the expression patterns of these checkpoint molecules were closely associated with treatment response and provided some useful direction when choosing chemotherapeutic drugs. These findings enhance our understanding of these checkpoints in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies.

BACKGROUND: Pembrolizumab shows robust antitumor activity and favorable safety in metastatic melanoma. KEYNOTE-151 evaluated pembrolizumab in Chinese patients, who have more aggressive melanoma subtypes than other populations. METHODS: Chinese patients aged ≥18years with advanced melanoma previously treated with one line of therapy received pembrolizumab 2 mg/kg every 3 weeks for 35 cycles or until confirmed disease progression, intolerable toxicity, or study withdrawal. Primary end points were objective response rate (ORR) per RECIST v1.1 by blinded independent central review and safety. Key secondary end points included duration of response (DOR) and progression-free survival (PFS) per RECIST v1.1 and overall survival (OS). RESULTS: Median age was 52 years (N=103); 37.9% had acral and 14.6% had mucosal melanoma. Median follow-up was 7.9months at data cutoff (December 27, 2017). ORR was 16.7% (95% CI, 10.0-25.3%) (1 complete, 16 partial responses). Disease control rate was 38.2%. ORR was 15.8% for acral, 13.3% for mucosal melanoma. Median DOR was 8.4months; 65.6% of patients had response duration ≥6months. Median PFS was 2.8months (95% CI, 2.7-3.5months); 6-month rate was 20.4%. Median OS was 12.1months (95% CI, 9.6months-not reached); 6-month rate, 75.7%; 12-month rate, 50.6%. Treatment-related AEs (TRAEs) occurred in 87 (84.5%) patients; 9 (8.7%) experienced grade 3/4 TRAE and 2 (1.9%) discontinued because of TRAE; none died. Two deaths occurred that were unrelated to treatment. CONCLUSIONS: Pembrolizumab was well tolerated and provided clinically meaningful antitumor activity as second-line therapy in Chinese patients with advanced melanoma.

We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR + TCR deficient human Tregs maintained both Treg phenotype and function in vitro . Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR + TCR deficient Tregs did not impair the function of these HLA-A2 + islets, whereas similarly engineered A2-CAR + TCR deficient CD4 + conventional T cells rejected the islets in less than 2 weeks. A2-CAR + TCR deficient Tregs delayed graft- versus -host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.