Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells.

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255-2262, 2016; Leidner et al in N Engl J Med, 386:2112-2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40 years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

Exploration of the relationship between intestinal flora changes and gut acute graft-versus-host disease after hematopoietic stem cell transplantation.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a life-threatening factor for post-hematopoietic stem cell transplantation (HSCT) patients. To investigate the relationship between intestinal flora changes and gut aGVHD after HSCT, we performed this cross-sectional study. METHODS: We selected children from our medical center from July 2016 to January 2017. Fifty-six samples from 42 patients and 6 samples from normal children met the study criteria and were analyzed. Fecal 16S RNA sequencing was completed before transplantation or on days 7, 28 or 100 post-transplantation. The intestinal infection and GVHD clinical data were retrospectively analyzed, and the survival risk factors were analyzed. Correlation analysis was performed with the feces bioinformatic data. RESULTS: The GVHD group alpha diversity was the lowest, which was significantly different than that of the non-diarrhea group (P value=0.032). A richer posttransplantation relative abundance of Moraxellaceae was conducive to survival, while that of Enterococcaceae and Alphaproteobacteria was not. Similarly, a rich relative abundance of Proteobacteria, Gammaproteobacteria and Odoribacteraceae in the intestinal flora before HSCT contributed to patient death thereafter. Regarding diarrhea, the GVHD group exhibited a richer Pasteurellales and Pasteurellaceae relative abundances, which showed strong correlations with diarrhea severity. Peptostreptococcaceae, Bifidobacteriales and Bifidobacteriaceae were richer in relative abundance in the intestinal infection group and correlated with pretransplant characteristics. CONCLUSIONS: The gut microbiota diversity was lowest when gut aGVHD occurred, which was consistent with the clinically higher mortality rate and greater treatment difficulty. Pasteurellaceae played an important role in gut aGVHD and diarrhea severity. Bifidobacteriaceae led to infectious diarrhea after HSCT. Specific bacteria were biomarkers for survival: Moraxellaceae, Enterococcaceae and Alphaproteobacteria from the intestinal flora after HSCT and Proteobacteria, Gammaproteobacteria and Odoribacteraceae before HSCT.

Unaccompanied mechanosensory domain mediates low expression of glycoprotein Ibα: implications for Bernard-Soulier syndrome.

BACKGROUND: Disruption of protein folding or inter-subunit interactions in the platelet glycoprotein (GP)Ib-IX complex leads to its abnormally low expression in the plasma membrane, the hallmark of Bernard-Soulier syndrome (BSS). OBJECTIVE: To discover the molecular mechanism by which GPIbα in the absence of GPIbß and GPIX subunits is targeted for rapid degradation. METHOD: The expression of GPIbα mutants with deletion or replacement of various domains were measured in transiently transfected Chinese hamster ovary cells. RESULTS: We report evidence to suggest that induction of the unfolded protein response by the unaccompanied mechanosensory domain (MSD) is a major factor for intracellular degradation and low expression of GPIbα. Removal of the MSD produced the first GPIbα variant that, even in the absence of GPIbß and GPIX, expressed at a level comparable to that of wild-type GPIbα in the GPIb-IX complex, while retaining its native ligand-binding activity. CONCLUSION: Our finding has important implications on the molecular pathogenesis of BSS and the function of the GPIb-IX complex.

Rapid identification of pathogens involved in pediatric osteoarticular infections by multiplex PCR.

BACKGROUND: Delays in the diagnosis of pediatric osteoarticular infections (OAIs) can cause associated acute complications or long-term morbidity. This study attempts to develop a multiplex PCR-based assay that can rapidly and accurately detect the main pathogens involved in pediatric OAIs, namely, methicillin-sensitive or methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa. METHODS: A set of four gene-specific primers suitable for use in a one-tube PCR assay was designed to detect four common pathogens involved in pediatric OAIs, namely, nuc for methicillin-sensitive Staphylococcus aureus, nuc and mecA for methicillin-resistant Staphylococcus aureus, spyM for Streptococcus pyogenes and orpI for Pseudomonas aeruginosa. The multiplex PCR was first evaluated with 39 isolated clinical strains and further with 41 specimens collected from patients suspected of having OAIs. RESULTS: Specific primer pairs were successfully designed, and the targeted genes were simultaneously amplified. The product sizes in the assay for nuc, mecA, spyM and oprI were 233, 158, 336 and 109 bp, respectively. Evaluation of the multiplex PCR with 39 isolated clinical strains and 41 specimens revealed 100% sensitivity and 100% specificity. The limit of detection of the multiplex PCR assay was approximately 1×103 CFU at the bacterial cell level. CONCLUSIONS: This newly developed multiplex PCR assay, without sequencing, enables a rapid and accurate diagnosis of the major bacterial species in children with OAIs and might serve as an additional diagnostic approach for urgent pathogen determination.